1. No category

IDENTIFICATION, CHARACTERIZATION AND
UTILITY OF PROSTATE-SPECIFIC ANTIGENBINDING PEPTIDES
Ping Wu
Department of Clinical Chemistry
Helsinki University Central Hospital
University of Helsinki
Helsinki, Finland
Academic Dissertation
To be publicly discussed with the permission of the Medical Faculty of the
University of Helsinki, in Auditorium 2, Biomedicum Helsinki, on December 20,
2004, at 12 noon.
Helsinki 2004
Supervised by
Professor Ulf-Håkan Stenman
University of Helsinki
Finland
Reviewed by
Docent Pirjo Laakkonen
University of Helsinki
Finland
and
Professor Kim Pettersson
University of Turku
Finland
Opponent
Docent Mathias Bergman
University of Helsinki
Finland
ISBN 952-91-8068-3 (paperback)
ISBN 952-10-2228-0 (PDF)
http://ethesis.helsinki.fi
Yliopistopaino
Helsinki 2004
TABLE OF CONTENTS
1. List of original publications.........................................................................................5
2. Abbreviations ................................................................................................................6
3. Introduction ...................................................................................................................7
4. Review of the literature................................................................................................8
4.1. Prostate ........................................................................................................................8
4.1.1. Anatomical structure ..................................................................................8
4.1.2. Cell types (histological structure).............................................................8
4.1.3. Ejaculate and secretory proteins in seminal fluid ..................................9
4.2. Prostate-specific antigen (PSA) ................................................................................9
4.2.1. Expression of PSA.....................................................................................10
4.2.2. Biochemistry and structure of PSA ........................................................10
4.2.3. Biological function of PSA .......................................................................12
4.2.4. Molecular forms of PSA in biological fluids .........................................12
4.2.5. Clinical use of PSA....................................................................................13
4.3. Prostate cancer..........................................................................................................14
4.3.1. Diagnosis ....................................................................................................14
4.3.2. Treatment ...................................................................................................14
4.4. Benign prostatic hyperplasia (BPH) ......................................................................15
4.5. Phage display methodology...................................................................................15
4.5.1. Biology and structure of bacteriophage.................................................15
4.5.2. Principles and applications of phage display .......................................17
4.6. Nuclear magnetic resonance (NMR) technology ................................................18
5. Aims of the study........................................................................................................20
6. Materials and methods...............................................................................................21
6.1. Samples......................................................................................................................21
6.2. Antibodies .................................................................................................................21
6.3. Phage display peptides ...........................................................................................21
6.3.1. Construction of phage display peptide libraries ..................................21
6.3.2. Selection of PSA-binding peptides by biopanning..............................22
6.4. Production of PSA-binding peptides as GST fusion proteins ...........................23
6.5. Synthesis of PSA-binding peptides and its derivatives......................................23
6.6. Immunoassays..........................................................................................................23
6.6.1. Time-resolved immunofluorometric assays (IFMAs)..........................23
6.6.2. Phage IFMA ...............................................................................................24
6.6.3. Immunopeptidometric assay (IPMA) ....................................................24
6.7. Measurement of enzymatic activity ......................................................................24
6.7.1. Effect of peptides on the enzymatic activity of PSA ............................24
6.7.2. Determination of proPSA activation ......................................................25
6.8. Chromatographic methods.....................................................................................25
6.8.1. Purification of PSA ....................................................................................25
6.8.2. Ion exchange chromatography ...............................................................25
6.8.3. Gel filtration...............................................................................................26
6.8.4. Immunoaffinity chromatography...........................................................26
6.8.5. Peptide affinity chromatography ...........................................................26
6.9. Electrophoresis .........................................................................................................26
6.10. Surface plasmon resonance ..................................................................................27
6.11. Nuclear magnetic resonance (NMR) method ....................................................27
7. Results...........................................................................................................................28
7.1. Identification of PSA-binding peptides (I) ...........................................................28
7.2. Peptide affinity chromatography (II) ....................................................................35
7.3. Immunopeptidometric assay---a new assay principle (III)................................37
7.4. Structural analysis of PSA-binding peptides (IV) ...............................................38
8. Discussion ....................................................................................................................40
8.1. Identification and characteristics of novel PSA-binding ligands......................40
8.2. Conformational and biochemical analyses of PSA-binding peptides..............41
8.3. Separation of enzymatically active and inactive PSA by
peptide affinity chromatography ..........................................................................42
8.4. Immunopeptidometric assay for enzymatically active PSA..............................42
9. Summary ......................................................................................................................43
10. Acknowledgments ....................................................................................................45
11. References ..................................................................................................................47
12. Original publications ................................................................................................59
4
1. LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original publications referred to in the text
by their Roman numerals (I-IV):
I) Wu P, Leinonen J, Koivunen E, Lankinen H, Stenman UH. Identification of
novel prostate–specific antigen-binding peptides modulating its enzyme activity.
Eur J Biochem 2000;267:6212-6220.
II) Wu P, Stenman UH, Pakkala, M, Närvänen A, Leinonen J. Separation of
enzymatically active and inactive prostate-specific antigen (PSA) by peptide
affinity chromatography. Prostate 2004;58:345-353.
III) Wu P, Zhu L, Stenman UH, Leinonen J. Immunopeptidometric assay for
enzymatically active prostate-specific antigen. Clin Chem 2004;50:125-129.
IV) Pakkala M, Jylhäsalmi A, Wu P, Leinonen J, Stenman UH, Santa H,
Vepsäläinen J, Peräkylä M, Närvänen A. Conformational and biochemical
analysis of the cyclic peptides which modulate serine protease activity. J Peptide
Sci 2004;10:439-447.
5
2. ABBREVIATIONS
A2M
D2-macroglobulin
aa
amino acid
ACT
D1-antichymotrypsin
API
D1-protease inhibitor
BPH
benign prostatic hyperplasia
GST
glutathione S-transferase
hK1
tissue kallikrein
hK2
human kallikrein 2
IFMA
immunofluorometric assay
IPMA
immunopeptidometric assay
kDa
kilodalton
MAb
monoclonal antibody
MW
molecular weight
NMR
nuclear magnetic resonance
PCa
prostate cancer
pI
isoelectric point
proPSA
proenzyme form of PSA
PSA
prostate-specific antigen
SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
Serpin
serine proteinase inhibitor
6
3. INTRODUCTION
Prostate cancer is the most common cancer and the second leading cause of
cancer mortality in males in industrialized countries (1). The disease is curable if
detected early and when localized in the prostate. However, a large proportion
of prostate cancers are diagnosed at clinically advanced stages (2). Prostate
cancer can be detected at an early stage by determination of prostate-specific
antigen (PSA) in serum and biopsy of men with elevated concentrations of PSA
(3).
PSA is a 33-kDa serine protease mainly produced by both normal and
malignant prostatic epithelial cells (4-6). Serum PSA is the most important
marker for diagnosis and monitoring of prostate cancer (3). However, the use of
PSA in early detection of prostate cancer is problematic due to the high falsepositive rate caused by benign prostatic hyperplasia (BPH) (7, 8). About 5-35% of
PSA in serum is in free form (9, 10); these forms comprise proPSA, enzymatically
active PSA, and internally cleaved, enzymatically inactive PSA (11-13). Some
studies have suggested that enzymatically active PSA and proPSA in serum are
preferentially secreted by prostate cancer cells, and the cleaved forms are more
specific to benign prostatic hyperplasia. Thus, determination of these PSA
variants can reduce the number of false-positive results in early diagnosis of
prostate cancer (14, 15).
The main biological function of PSA is liquefaction of the seminal gel formed
after ejaculation by proteolysis of semenogelins (16, 17). PSA has been proposed
to inhibit prostate cancer growth and metastasis by proteolytic cleavage of
plasminogen, generating angiostatin-like fragments (18-20). Angiostatin is an
inhibitor of angiogenesis, a process necessary for tumor growth and metastasis.
Thus, ligands binding to PSA and modulating its enzymatic activity are
potentially useful for treatment of prostate cancer.
Phage display is a powerful approach for selection of novel ligands for
various target molecules such as enzymes (21-23), antibodies (24), and receptors
(25, 26). Phage peptide libraries offer a means of searching for ligands possessing
unique binding specificities.
7
4. REVIEW OF THE LITERATURE
4.1. Prostate
4.1.1. Anatomical Structure
The human prostate is part of the male reproductive system and the accessory
genital glands, along with the seminal vesicles and Cowper´s glands. Androgen
action plays a central role in the physiological and pathological processes of the
prostate (27, 28). The prostate of a normal adult male weighs 20 ± 6 g and is
located immediately below the base of the bladder surrounding the proximal
portion of the urethra (29, 30).
The prostate can anatomically be divided into five lobes: anterior lobe,
posterior lobe, median lobe, and two lateral lobes. A capsule of collagenous and
elastic fibers and smooth muscle tissue encloses the prostate (31). For clinical
purposes, the prostate is divided into three zones: the central zone, peripheral
zone, and transition zone (Fig. 1) (31, 32). The peripheral zone is much larger
than the central zone, and together they constitute ~95% of the prostatic
glandular structure. The transition zone makes up ~5% of the glandular structure
(32, 33), and it is the part of the prostate that is enlarged in prostatic hyperplasia
(34). The peripheral zone is the most common site of prostate cancer (34).
4.1.2. Cell types (histological structure)
The prostate contains fibromuscular (30%) and glandular (about 70%)
components. Histologically, the ductal glands consist of epithelial cells
surrounded by stromal tissue (31, 35). The prostatic epithelium consists of three
types of cells: columnar secretory epithelial cells, basal cells, and neuroendocrine
cells (33, 35). The columnar secretory epithelial cells produce PSA, human
kallikrein 2 (hK2), prostatic acid phosphatase (PAP), and other secreted proteins.
Basal cells are important in the histological diagnosis of prostate cancer because
prostate cancer acini lack basal cells. Neuroendocrine cells contain somatostatin,
calcitonin, and other hormones scattered among secretory cells (36, 37).
The stromal cells consist of fibroblasts, capillary and lymphatic endothelial
cells, smooth muscle cells, neuroendocrine cells, and axons (35). Stromal cells
serve as a matrix for the epithelium and contribute a variety of growth factors
that regulate secretion, proliferation, differentiation, and apoptosis of epithelial
cells (33, 38).
8
Fig. 1. Zones of the prostate.
Reprinted from Walsh PC, Retik AB, Stamey TA, Vaughan DJ. Campbell´s Urology.
W.B. Saunders, Sixth ed, 1:344, 1992, with permission from Elsevier (Ref. 31).
4.1.3. Ejaculate and secretory proteins in seminal fluid
The volume of normal human ejaculate ranges from 2 to 6 mL, the average
volume being 3 mL (31). The ejaculate is composed of two main components,
spermatozoa and seminal plasma. Spermatozoa account for less than 1% of the
ejaculate volume. The prostatic fluid mixes with components from secretions of
other male sex accessory glands to form seminal plasma. The major contribution
originates from the seminal vesicles (1.5-2 mL), the prostate (0.5 mL), Cowper´s
gland and the glands of Littre (0.1-0.2 mL) (39-42). The predominant prostatic
secretory proteins are PSA, PAP, prostate-specific protein 94 (PSP94 or EMSP)
and hK2. PSA is the most abundant protein secreted by the prostate (43-45).
4.2. Prostate-specific antigen (PSA)
PSA was first purified from prostatic tissue and shown to be prostate-specific in
1979 (4). One year later, it was demonstrated to be present in serum of patients
with prostate cancer (46, 47).
9
4.2.1. Expression of PSA
PSA is produced by normal and malignant prostatic epithelial cells, and its
expression is stimulated by androgens (48). PSA occurs at very high
concentrations, ranging from 0.5-2 mg/mL, in human seminal plasma (49). PSA is
quite organ-specific but it has been shown to be expressed at low concentrations
in other human tissues and body fluids, e.g. in the periurethral glands, the anal
gland, and breast tissue of males and females (50-53). PSA can occasionally be
found in other cancer types, such as adrenal, kidney, lung, and colon cancers,
and it has also been used as a prognostic marker for breast cancer (54-57). In
ultrasensitive immunoassays, PSA can be detected at very low concentrations in
female serum and other body fluids, including amniotic fluid, breast fluid, cyst
fluid, and nipple aspirate fluid (58-60).
In healthy men, PSA is mainly secreted into seminal fluid and only small
amounts leak from the normal prostate into circulation; thus, serum
concentrations are about one million-fold lower than those in seminal fluid (61).
In prostatic disease, the concentrations of serum PSA are elevated due to
increased leakage of PSA into circulation (61).
4.2.2. Biochemistry and structure of PSA
The human kallikreins (hK) are encoded by 15 structurally related genes (KLK)
that colocalize to chromosome 19q13.4 (long arm of chromosome 19) (62-64). PSA
is a chymotrypsin-like serine protease belonging to the human kallikrein family
(62-64). It is encoded by a gene that is clustered within a 300-kb region on
chromosome 19q13.4, together with hK2, tissue kallikrein (hK1), and 12 other
recently characterized kallikrein genes (Table 1) (63-66). All members of the
kallikrein family have five coding exons and display considerable sequence
homologies (40-79%) at the DNA and amino acid levels. The best-known
members are hK1, hK2, and PSA (hK3). PSA and hK2 are the most closely
related, with 79% identity in amino acid sequence, while hK1 has 62% identity to
PSA (67).
The enzymatic activity of PSA is chymotrypsin-like but very restricted, and it
preferentially hydrolyzes peptide bonds at the carboxy-terminus of the
hydrophobic residues tyrosine and leucine (68). Several residues surrounding
these preferred P1 residues play an important role in determining the substrate
specificity (17, 69-71). Most studies on the enzymatic activity of PSA have
utilized synthetic peptide substrates for chymotrypsin. Recently, peptide
substrates based on the cleavage sites of PSA on semenogelines I and II have
10
been developed (17, 70, 72). Substrate phage display has also been used to study
the enzymatic activity of PSA (71).
PSA contains the catalytic triad characteristic of serine proteases. Residues
His-41, Asp-96, and Ser-189 are located in typical sites of the active pocket (73,
74). Ser-183, which is located at the bottom of the active site, is crucial for the
specificity of PSA (75).
Table 1. Members of the human Kallikrein family.
Gene
KLK1
KLK2
KLK3
KLK4
KLK5
KLK6
KLK7
KLK8
KLK9
KLK10
KLK11
KLK12
KLK13
KLK14
KLK15
Protein (symbols)
hK1
hK2
hK3
hK4
hK5
hK6
hK7
hK8
hK9
hK10
hK11
hK12
hK13
hK14
hK15
Protein (names)
tissue kallikrein
human kallikrein 2
prostate-specific antigen
prostase
human kallikrein 5
protease M
human kallikrein 7
neuropsin
human kallikrein 9
human kallikrein 10
human kallikrein 11
human kallikrein 12
human kallikrein 13
human kallikrein 14
prostinogen
PSA is a single-chain glycoprotein. The structure deduced from the cDNA
sequence shows that PSA is synthesized as a 261 amino acid (aa) prepropeptide
comprising a 17-aa signal peptide, followed by a 7-aa propeptide and a 237-aa
mature, enzymatically active protein (68, 76). The signal peptide is removed
before secretion. ProPSA containing the activation peptide is enzymatically
inactive and can be activated into the mature form by proteolytic cleavage with
trypsin, hK2, hK4, and prostin (77-81). Activation with trypsin is the most
efficient, followed by hK4 or prostin, and finally hK2 (77, 80-82). In seminal fluid,
PSA occurs in the intact, enzymatically active form, but also in internally cleaved,
inactive forms (nicked PSA) (83). The internal cleavage sites in the peptide
backbone are at Arg-85, Lys-145 and Lys-182. They disrupt the conformation of
PSA, causing loss of catalytic activity (62, 83-85).
About 70% of PSA purified from seminal fluid is intact and enzymatically
active, while 30% is nicked (83, 84). The average molecular weight (MW) of
11
native PSA determined by mass spectrometry is 28430 (86) and the relative
molecular mass determined by SDS-PAGE or gel filtration is about 33 kDa (5, 49,
86). The lymph node cancer of the prostate (LNCaP) cell line (87) mainly secretes
PSA as an inactive proenzyme (82). Väisanen et al. found that about half of PSA
consisted of a one-chain, mature, and enzymatically inactive form when LNCaP
cells were grown in a medium containing fetal bovine serum (88). However, this
one-chain mature PSA was enzymatically active when the LNCaP cells were
grown in a medium without serum (88). The proenzyme form of PSA has also
been produced as a recombinant protein (78, 89, 90). No proPSA has been
detected in seminal fluid (91).
4.2.3. Biological function of PSA
The main biological function of PSA is liquefaction of the seminal gel formed
after ejaculation by proteolytic cleavage of semenogelin I and II, which are the
major constituents of the seminal gel (16). Some studies have suggested that PSA
plays a role in both inhibition or promotion of prostate cancer invasion and
metastasis. PSA may inhibit cell growth and angiogenesis by generating
angiostatin-like fragments from plasminogen (18-20), and it may also induce
apoptosis (92). Other experiments suggest that PSA activates the urokinase-type
plasminogen activator, which is thought to be involved in tumor invasion and
metastasis (93), and affects tumor spread by proteolytic modulation of cell
adhesion receptors (94). Furthermore, PSA has been found to cleave insulin-like
growth factor binding protein 3 (IGFBP-3) and insulin-like growth factor binding
protein 4 (IGFBP-4), causing release of active insulin-like growth factor 1 (IGF-I),
which could enhance tumor growth (95, 96). PSA also cleaves fibronectin and
laminin (97), and activates latent TGF-beta (94), and by these mechanisms it may
mediate progression of prostate cancer.
4.2.4. Molecular forms of PSA in biological fluids
In serum, the majority (50-90%) of the immunoreactive PSA consists of a complex
with D1-antichymotrypsin (PSA-ACT); minor parts occur in complex with other
protease inhibitors, while about 5-35% of PSA is in free form (9, 10, 98).
Free PSA in serum consists of different isoforms, mainly internally cleaved
forms and proPSA (11, 99-101), but a small enzymatically active fraction has also
been detected (13). ProPSA lacks enzymatic activity and does not form
complexes with protease inhibitors (78, 88). In addition, truncated forms of
proPSA occur in serum from healthy men and patients with prostate cancer (15).
A high proportion of nicked PSA has been found in BPH tissue and seminal
plasma (102, 103). Serum from PCa and BPH patients also contains nicked PSA
12
(11, 101); the proportion of this form is in fact higher in BPH than in cancer (99,
100). Part of this PSA is internally cleaved after Lys-145 and Lys-182, and it is
called BPSA (103, 104). Some assays detecting free PSA variants have been
described. One assay uses a MAb that does not recognize PSA cleaved after Lys145 but binds to intact, mature PSA (105). Another assay that specifically detects
internally cleaved PSA (after Lys-182) has recently been reported (104).
Epithelial cells of the normal prostate secrete PSA into the prostatic glandular
ducts. PSA reaches the circulation only by leaking backwards into the
extracellular fluid and then diffusing into blood circulation (61). However, when
prostate cancer looses tissue architecture, polarization, and the connection to the
prostatic ducts, PSA may be released directly into extracellular fluid and
circulation in a more active form than when leaking out from BPH tissue (61, 98).
This could explain why the proportion of PSA occurring in complex with ACT is
increased in prostate cancer serum. Niemelä et al. used an enzymatic assay to
show that about 3% of PSA in serum from PCa patients occurs in an
enzymatically active form (13). Noldus et al. analyzed free PSA from serum of
PCa patients and found both intact (mature) and internally cleaved forms. The
amino terminal sequence showed that the N-terminus is identical to that of free
PSA from seminal fluid (100).
4.2.5. Clinical use of PSA
PSA was first detected in the serum of PCa patients In 1980, and after the
pioneering studies of Stamey in 1987, measurement of serum PSA became a
widely used marker for early detection, screening, and monitoring of PCa (3, 7).
PSA is not, however, a perfect tumor marker because PCa, BPH, and prostatitis
can all cause increased serum PSA concentrations. Manipulation of the prostate,
e.g. in cystoscopy and prostate biopsy, can also increase the concentrations of
serum PSA. In apparently healthy men, the serum PSA level is below 4 µg/L in
90%, 4-10 µg/L in 8%, and over 10 µg/L in 2% (106-108). A serum PSA below 4
µg/L is traditionally considered to indicate a low risk for PCa. When serum PSA
is in the range 4-10 µg/L, about 25-30% of these men have cancer detected by
biopsy (7, 109). Men with serum PSA levels above 10 µg/L have a 40-50%
likelihood of PCa (109). However, PCa has also been detected in 20-30% of men
with a PSA of 3-4 µg/L (107, 110, 111). The proportion of PSA-ACT is higher and
that of free PSA is lower in serum of PCa patients than in BPH patients (2, 7, 9,
10), and by assaying the proportion of either PSA-ACT or free PSA in relation to
total PSA, about 20-30% of the false-positive results in BPH can be avoided (9). In
spite of this, approximately two-thirds of the elevated results are still falsepositives (112). In addition, about 50% of men over 60 years of age have
microscopic PCa. Thus, only 5-10% of the cancer is detected by PSA-based
13
screening (61, 98).
4.3. Prostate cancer
More than 95% of all prostate cancers are adenocarcinomas arising from the
epithelium of the acini and terminal ducts. Most of the remainder are transitional
cell carcinomas (31).
4.3.1. Diagnosis
PCa is mainly located in the peripheral zone of prostate, with a small portion
occuring in the central zone (34, 113). About 30-50% of men older than 50 years
have a latent PCa, as assessed by postmortem autopsy (114).
PCa is classified according to various grading systems. The Gleason grading
system is widely used for pathological classification (115, 116). The TNM (tumor,
node, metastasis) system is used to further classify the stage of cancer (117, 118).
Usually, PCa causes clinical symptoms only when the cancer has spread to
surrounding tissues or metastasized to lymph nodes or other organs of the body.
Development of the serum PSA assay revolutionized the diagnosis and
management of PCa. Diagnosis is currently based on the combined use of digital
rectal examination (DRE), PSA assay, transrectal ultrasonography (TRUS), and
transrectal ultrasound-guided biopsies (119, 120).
4.3.2. Treatment
Treatment strategies for PCa depend on the age and physical condition of the
patient, the tumor stage, and the histopathological grade. If the cancer is
localized and the general condition of the patient is good, radical prostatectomy
or radiotherapy is the primary treatment (121-123). Alternatively, a watchful
waiting is a viable option for some patients (2). In patients with local invasion,
positive lymph nodes, or distant metastases, hormonal therapy is typically used.
This approach slows tumor growth and reduces tumor volume but is not
curative (124-126). Radiotherapy can also be used for local therapy of hormoneresistant advanced disease. Recently, some novel therapeutic approaches, such as
gene therapy (127), tumor-targeting with phage display peptides to selectively
deliver cytotoxic drugs (128), and enzymatic activation of prodrugs (129), have
been tested in experimental models.
14
4.4. Benign prostatic hyperplasia (BPH)
BPH is a very common disease in elderly men and affecting the same agegroup
as PCa. The two diseases often co-exist. Pathophysiologically, enlarged
hyperplastic nodules in the prostatic transition zone cause an increase in prostate
volume. This compresses the prostatic urethra and elevates urethral pressure.
However, prostatic size does not correlate with the degree of clinical symptoms.
Options for treatment include transurethral resection (TURP), open surgery, and
drug therapy.
4.5. Phage display methodology
Phage display is a molecular diversity technology, which was first described by
George Smith in 1985 (130). It presents a genetically well-defined system and has
been applied to a broad spectrum of biological studies. Selection of peptides or
proteins from phage display libraries is now an effective approach in searching
for novel ligands for various target molecules. Phage display has recently been
used in vivo for exploring peptides that home specifically to tumor blood vessels,
lymphatic vessels, or cancer cells to develop targeted drug delivery tools for
tumors (23, 131-134).
4.5.1. Biology and structure of bacteriophage
Filamentous bacteriophage or phage comprise a large family of bacteriophage
infecting different Gram-negative bacteria without lysing the host cells (135). The
most widely applied phage are M13, fl, and fd. These infect Escherichia coli (E.
coli) male strains by attaching of the end of the phage (pIII) to the F pilus on the
bacterial surface (sex pili encoded by the F episome) (136). Phage have a singlestranded, covalently closed DNA genome, which is around 6400 nucleotides long
and comprises 11 genes (Fig. 2) (137). The single-stranded DNA (ssDNA) is
extruded through the membrane and is covered by capsid proteins. Bacterial
enzymes synthesize the complementary strand and convert the infecting phage
DNA into a supercoiled, double-stranded replicative form (RF) (137). This RF
undergoes rolling circle replication to create a single-stranded DNA and also
serves as a template for the synthesis of phage proteins (138).
Phage particles are about 6-7 nm wide, 900-2000 nm long, and have a mass of
approximately 16.3 MDa (Fig. 3) (137, 139). Three of the 11 genes in phage are
required for DNA synthesis (products of genes II, V, and X) (Table 2), and five
are virion structural proteins (those encoded by genes III, VI, VII, VIII, and IX).
Two genes (I and IV) are involved in phage morphogenesis (137, 140). The
products of structural genes VII and IX (pVII and pIX) are incorporated at the
15
end of the virion that is extruded first (C-terminal end) and gene V proteins (pV)
are removed from the DNA and shed back into the cytoplasm, being replaced by
molecules of the major coat protein (pVIII). The products of genes III and VI
(pIII and pVI) are incorporated at the other end (N-terminal) of the viral particle,
and the assembled phage is released into the medium (136, 137).
Fig. 2. Genome of the f1 bacteriophage.
The single-stranded DNA contains 11
genes and has 6407 nucleotides, which
are numbered from the unique HindII site
located in gene II (represented by 0). IG,
intergenic region; PS, packaging signal;
(+/-), the position of the origin of
replication
of
the
viral
and
complementary DNA strands; arrows
indicate the location of the overlapping
genes X and XI. Reprinted from Kay BK,
Winter J, McCafferty J. Phage Display of
Peptides and Proteins. A Laboratory Manual, 5, Academic Press, 1996, with permission
from Elsevier (Ref. 137).
Fig. 3. The Ff bacteriophage particle.
Schematic representation of the phage particle showing location of the capsid proteins
and orientation of the DNA. Reprinted from Kay BK, Winter J, McCafferty J. Phage
Display of Peptides and Proteins. A Laboratory Manual, 9, Academic Press, 1996, with
permission from Elsevier (Ref. 137).
There are two important coat proteins on the phage. The major coat protein is
pVIII, a 50-aa residue protein of which the phage expresses about 2700 copies.
The gene III protein (pIII) contains 406 amino acids and expresses 3-5 copies on
16
each phage (140).
Table 2. Genes and gene products of f1 bacteriophage.
Gene
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
No. of
amino acids
348
410
406
405
87
112
33
50
32
111
108
MW
Function
39,502
46,137
42,522
43,476
9,682
12,342
3,599
5,235
3,650
12,672
12,424
Assembly
DNA replication
Minor capsid protein
Assembly
Binding ssDNA
Minor capsid protein
Minor capsid protein
Major capsid protein
Minor capsid protein
DNA replication
Assembly
Function
group
3
1
2
3
1
2
2
2
2
1
3
Gene function group 1 encodes proteins required for DNA replication; gene function
group 2 encodes proteins which make up the capsid of the phage; gene function group 3
encodes proteins involved in the membrane-associated assembly of the phage. MW,
molecular weight. Adapted from Kay BK, Winter J, McCafferty J. Phage Display of
Peptides and Proteins. A Laboratory Manual, Academic Press, 1996 (Ref. 137).
4.5.2. Principles and applications of phage display
Phage display is an effective method for selection of novel ligands for target
molecules. Foreign peptides or proteins are displayed on the surface of the
phage by inserting the displayed genes (DNA fragments or synthetic
oligonucleotides) between the signal peptide and the structural gene of the gene
coding for the major (pVIII) or minor (pIII) coat proteins (Fig. 3). This facilitates
linking the phenotype of the phage to its genotype (130).
Peptides displayed on pVIII have the benefit of more of their copies being
displayed per phage (about 2700 copies), but the length of the inserts is restricted
to 6-8 amino acids (141). Inserts fused to pIII can code for peptides longer than
those fused to pVIII, and inserts comprising up to 15 amino acids do not disturb
the function of the phage coat proteins (142, 143). To display larger size peptides
or proteins, construction of hybrid viral vectors are needed (144).
The key steps of applying phage display are as follows (145-148) (Fig. 4):
1) DNA technology is used to build phage display libraries. Fragments of DNA
or synthetic oligonucleotides encoding the proteins or peptides are inserted at an
17
engineered restriction site in phage gene III or VIII.
2) The libraries are screened with target molecules of interest to select specific
phage-displayed peptides or proteins. This step is termed “biopanning”.
3) The phage bound to target molecules can be eluted and amplified by infecting
the bacteria with phage and again selecting for binding to the same target
molecule.
4) Sequences of the selected peptides or proteins can be deduced by DNA
sequencing of selected individual phage clones released from bacterial host cells.
Inserting displayed gene in phage gene III or VIII
Screening phage library with target molecule
Target
molecule
Target
molecule
Target
molecule
Removing nonspecific phage by washing
Target
molecule
Eluting specifically bound phage
Amplifying eluted phage
Determining the sequence of selected phage
Fig. 4. Illustration of the phage display technology.
Adopted from Koivunen E, Restel BH, Rajotte D, Lahdenranta J,
Hagedorn M, Arap W, et al. Integrin-binding peptides derived from
phage display libraries. Methods Mol Biol 1999;129:3-17 (Ref. 148).
The main applications of phage display are as follows:
1) To display natural peptides or proteins for mapping epitopes of antibodies (24,
149).
2) To display synthetic random peptides and proteins to search for novel ligands
for target molecules (25, 71, 150).
3) To display synthetic random peptides to identify ligands that home
specifically to tumor blood vessels, lymphatic vessels, and cancer tissues in order
to develop targeted therapy (23, 128, 131, 134).
4) To display proteins for evolution and improvement of protein function (151).
5) To generate potent and novel antibodies (152).
4.6. Nuclear magnetic resonance (NMR) technology
Nuclear magnetic resonance (NMR) is a powerful technology for analyzing the
structure and dynamics of molecules. It can provide structural information on
18
molecules at atomic resolution and as well as information on dynamics,
conformational equilibria, folding, and intra- and intermolecular interactions
(153, 154). NMR experiments are performed on a NMR spectrometer consisting
of two main components: a high field superconductive magnet that produces an
extremely homogeneous, strong static magnetic field and a console that can
record signals from the sample and generate electromagnetic waves in any
desired combination (155).
The basis of NMR experiments is a quantum mechanical property of the
nucleus, the spin. Usually, the nuclei of interest carry a spin of 1/2 that allows
only two different spin states; these are often referred to as “spin up” and “spin
down”. Associated with the spin is a magnetic moment, which can be interpreted
as a magnetic dipole (153).
Hydrogen nuclei (protons) are the most sensitive type of nuclei. Analysis of
biological molecules starts with large proton magnetization and transfer of the
signal via heteronuclei (e.g. carbon or nitrogen) back to protons to record the
NMR signal (free induction decay) with maximal sensitivity. The proton offers
the best sensitivity, hence constituting the preferred nucleus for detection of the
NMR signal.
In NMR experiments, each tested nucleus produces an individual signal
(resonance line) in the spectrum, and spectra of a biological molecule contain
hundreds or even thousands of resonance lines. By using conventional 1-D NMR,
it is not possible to analyze biological molecules due to the vast number of
resonance lines. The foundation of structural NMR studies is a high-quality
NMR spectra recorded with good sensitivity and spectral resolution.
Multidimensional NMR spectra provide both increased resolution and
correlations that are easy to analyze. Nowadays, NMR experiments with
biological molecules are often measured in two or three dimensions. However,
molecular structures larger than 30 kDa are hard to study by NMR (156, 157).
NMR measurements are usually carried out in solution under nearphysiological conditions. The key steps of NMR structure determination are as
follows:
1) Preparation of the protein solution.
2) NMR measurements.
3) Assignment of NMR signals to individual atoms in the molecule.
4) Identification of conformational constraints.
5) Calculation of the 3-D structure based on the basis of the constraints, which
are determined from experimental results.
19
5. AIMS OF THE STUDY
The purpose of this study was to utilize phage display to identify novel PSAbinding peptides and to use these as tools for developing new diagnostic
methods for prostate cancer.
Specific aims were as follows:
1) To identify novel binding ligands for PSA using phage display.
2) To use PSA-binding peptides for isolation of enzymatically active PSA.
3) To use PSA-binding peptides as agents for developing new diagnostic assays
for determination of enzymatically active forms of PSA in circulation.
4) To characterize the peptide structures required for binding to PSA.
20
CA, USA).
6.4. Production of PSA-binding peptides as GST fusion proteins
Single-stranded phage DNA was purified and the insert region was amplified by
PCR (160). The amplified DNA was isolated and subcloned between the BamHI
and EcoRI sites of the PGEX-2TK vector (Amersham Pharmacia Biotech, Finland).
Recombinants were verified by DNA sequencing. The glutathione S-transferase
(GST) fusion proteins were expressed in E. coli BL 21 cells and purified by
glutathione affinity chromatography (Amersham Pharmacia Biotech) as
described elsewhere (161). Peptides were released from the GST fusion partner
by thrombin cleavage (162).
6.5. Synthesis of PSA-binding peptides and its derivatives
PSA-binding peptides and various derivatives were chemically synthesized on
the PerSeptive 9050 Plus peptide synthesizer using Fmoc strategy with
TBTU/DIPEA as the coupling reagent and NovaSyn TGA with 4hydroxymethylphenoxyacetic acid linker as the solid phase (Novabiochem,
Läufelfingen, Switzerland). Side-chain protecting groups were used during
synthesis. Acetamidomethyl (Acm) was used to facilitate cyclization in the sidechains of Cys(1) in peptide B-2 and Cys(5) and Cys(10) in peptide C-4.
For coupling to CH-Sepharose, the peptides were modified by adding a
spacer sequence of alternating Lys and Ser residues connected to the amino
terminus of the peptide via an amino caproic acid (aca) bridge. The structure of
the peptides with the spacer was SKSKSKS-aca-CVFAHNYDYLVC (B-2) and
SKSKSKS-aca-CVAYCIEHHCWTC (C-4). Peptides with cysteines (Acm) were
cyclized by using the Iodine method (163).
Alanine replacement sets and other modified peptides were synthesized with
the Apex 396 DC multiple peptide synthesizer (Advanced ChemTech, Louisville,
KY, USA). In alanine replacement sets, all amino acids except cysteines were
replaced one by one. The same synthesis method and amino acids were used as
described above.
6.6. Immunoassays
6.6.1. Time-resolved immunofluorometric assays (IFMAs)
A monoclonal antibody (MAb) was coated onto microtitration wells and the
antibodies used as tracers were labeled with an europium (Eu3+) chelate (164). In
23
the assays, 25 µL of standard or sample and 200 µL of assay buffer [50 mM TrisHCl, pH 7.7, 150 mM NaCl, 33.3 µM bovine serum albumin (BSA), 1 µM bovine
globulin] were added to the wells. For assaying chromatographic fractions, a
sample volume of 200 µL (together with 25 µL of IFMA assay buffer with a 10fold BSA concentration) was used. After incubation, the wells were washed
(washing buffer: 150 mM NaCl, 7.7 mM NaN3, 0.2 g/L Tween 20) and filled with
200 µL of assay buffer containing Eu3+-labeled antibody. After incubation and
washing, 200 µL of enhancement solution (PerkinElmer Life Science, Wallac,
Finland) was added, and the fluorescence was measured with a Victor 1420
multilabel counter (Perkin-Elmer Life Science, Wallac).
6.6.2. Phage IFMA
The binding of individual phage clones to PSA was tested by phage IFMA. PSA
was added to MAb 5E4-coated wells for 1 h. After washing, individual phage
clone in 200 µL of assay buffer was added. After incubation for 1 h, the wells
were washed and filled with 200 µL of assay buffer containing 50 ng of Eu3+labeled anti-phage monoclonal antibody (P. Saviranta, University of Turku,
Finland). After further incubation for 60 min, the wells were washed four times,
enhancement solution was added, and fluorescence was measured.
6.6.3. Immunopeptidometric assay (IPMA)
PSA purified from seminal fluid, serum samples or proPSA before and after
activation with trypsin were used to study the reactivity of PSA-binding peptides
in the IPMA. Twenty-five microliters of samples or calibrators (enzymatically
active PSA) and 200 µL of assay buffer were added to MAb 5E4-coated wells and
incubated for 1 h. After incubation, the wells were washed and 1 µg of GSTpeptide and 200 µL of assay buffer were added. After incubation for 60 min, the
wells were washed and 50 ng of Eu3+-labeled anti-GST antibody (Amersham
Pharmacia Biotech) and 200 µL of assay buffer were added and incubation was
continued for 60 min. The wells were washed four times, enhancement solution
was added, and after 5 min the fluorescence was measured.
6.7. Measurement of enzymatic activity
6.7.1. Effect of peptides on the enzymatic activity of PSA
The effect of peptides on the enzymatic activity of PSA was assayed in the
absence or presence of Zn2+ by using the chromogenic substrate S-2586
(Chromogenix, Mölndal, Sweden). PSA was incubated with a 1- to 100-fold
24
molar excess of GST-peptide fusion proteins in Tris-buffered saline (TBS)
containing 0.5 g/L BSA for 1 h at 22oC. After addition of substrate to a final
concentration of 0.2 mmol/L, the absorbance was monitored at 5-min intervals at
405 nm. The effect of the peptides on the enzymatic activity of other proteinases
was also tested using the substrate S-2586 for chymotrypsin and cathepsin G, and
S-2222 (Chromogenix) for trypsin (165).
6.7.2. Determination of proPSA activation
Purified proPSA was incubated in a MAb 5E4-coated well with slow shaking for
1 h at 22oC. After washing two times, bound proPSA was activated by addition
of bovine trypsin (Sigma Chemical, St. Louis MO, USA) (2 nmol/L) in 200 µL
buffer (0.5% BSA/TBS) and incubated for 20 min at 22oC. After washing, a
fluorescent peptide substrate (Enzyme Systems Products, Livermore, CA, USA)
was added to a final concentration of 400 µmol/L, and fluorescence (excitation
wavelength at 355 and emission wavelength at 460 nm) was monitored for 120
min in a Victor1420 multilabel counter (Perkin-Elmer Life Science) (13).
6.8. Chromatographic methods
6.8.1. Purification of PSA
Seminal plasma was precipitated with ammonium sulfate at 25% and 70%
saturation. After dialyzing the precipitate at 70% saturation against 50 mmol/L
Tris buffer, it was applied to an immunoaffinity column containing MAb 5E4.
The column was washed and PSA was eluted with 0.1% trifluoroacetic acid. PSA
isoenzymes were further separated by anion exchange chromatography on a
Resource Q column (Amersham Pharmacia Biotech).
ProPSA was purified from the LNCaP cell culture medium using an
immunoaffinity column with PSA-MAb 4G10, followed by cation exchange
chromatography on a Resource S column (Amersham Pharmacia Biotech). The
purified proPSA was activated by treatment with bovine trypsin (Sigma) as
described (77).
6.8.2. Ion exchange chromatography
The isoenzymes (A-E) of PSA were further separated on a Resource Q column
(Amersham Pharmacia Biotech). Immunoaffinity-purified PSA was dialyzed
against 10 mmol/L Tris-HCl buffer, pH 8.7 (buffer A), and applied to the
Resource Q column. PSA isoenzymes were eluted with a linear gradient (0-100%)
of buffer A and buffer B (buffer A containing 0.3 mol/L NaCl). For purification of
25
proPSA, immunoaffinity-purified LNCaP-PSA was dialyzed against 50 mmol/L
phosphate buffer, pH 5.6 (buffer A), and applied to a Resource S column
(Amersham Pharmacia Biotech). ProPSA was eluted with a linear gradient (0100%) of buffer A and buffer B (buffer A containing 0.5 mol/L NaCl).
6.8.3. Gel filtration
Gel filtration was performed on a Superdex-200 HR 10/30 column or a 1.6x60 cm
Superdex-200 column (Amersham Pharmacia Biotech) using 50 mmol/L sodium
phosphate buffer, pH 7.4, containing 150 mmol/L NaCl and a flow rate of 15 or
30 mL/h. The column was calibrated using IgG (160 kDa) and BSA (67 kDa) as
molecular size markers.
6.8.4. Immunoaffinity chromatography
Affinity columns were prepared by coupling MAb 5E4 or MAb 4G10 (free PSAspecific antibody) to CNBr-activated Sepharose (Amersham Pharmacia Biotech).
After the application of the samples, the column was washed with 50 mmol/L
Tris-HCl containing 0.5 mol/L NaCl. PSA was eluted with 0.1% trifluoroacetic
acid (TFA) in water, and neutralized with 1 mol/L Tris base.
6.8.5. Peptide affinity chromatography
Peptides B-2 and C-4 with a spacer sequence were coupled to an activated CHSepharose gel (Amersham Pharmacia Biotech). After incubation at 4oC for 12 h,
the remaining active groups were blocked by adding 0.1 M Tris-HCl, pH 8, for 1
h. The peptide affinity column was equilibrated with 10 mmol/L Tris-HCl, pH
7.8, or with the same buffer containing 100 µmol/L ZnCl2 (binding buffers).
Partially purified (ammonium sulfate-precipitated), immunoaffinity-purified
PSA, purified LNCaP-proPSA before and after trypsin activation, or PEGprecipitated serum were dialyzed against binding buffer and applied to the
peptide affinity column at a flow rate of 0.5 mL/min. After washing with 15-25
bed volumes of 10 mmol/L Tris-HCL, bound PSA was eluted with 10 mmol/L
acetic acid.
6.9. Electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was
performed under reducing and nonreducing conditions on homogeneous 15% or
20% polyacrylamide gels with the PhastSystem (Amersham Pharmacia Biotech).
Isoelectric focusing (IEF) was performed with the PhastSystem (Amersham
26
Pharmacia Biotech) using a gel with the pH range 5-8. Proteins separated by
SDS-PAGE and IEF were detected by silver staining according to the instructions
of the manufacturer.
6.10. Surface plasmon resonance
The binding kinetics of selected peptides to PSA was studied by surface plasmon
resonance (166, 167) on a BIAcore 2000™ instrument (Biacore AB, Uppsala,
Sweden). PSA was immobilized to the solid phase by MAb H117, which binds to
the same epitope as MAb 5E4 (168, 169). The capture antibody was covalently
coupled onto the surface of the CM5 sensor chip. PSA in PBS was injected for 4
min at a flow rate of 5 µL/min. After washing, peptides as GST fusion proteins
were injected at various concentrations for 1 min at a flow rate of 20 µL/min. To
characterize the effect of Zn2+ on the affinity of the peptides, the sensorgrams
were recorded for a constant amount of peptide in the presence of Zn2+ (0-30
µmol/L) both in the running and sample buffer. PSA-binding to either chemically
activated/deactivated blank surface or MAb H117 alone was subtracted as a
nonspecific interaction. Regeneration after each measurement cycle was done
with 3 x 0.5 min injections of 10 mmol/L HCl. Binding data were analyzed using
BIAEVALUATION software (Biacore AB).
6.11. Nuclear magnetic resonance (NMR) method
The peptides were dissolved in DMSO-d6. All spectra were recorded at 300-320
K on a Bruker Avance 500 NMR spectrometer (Bruker, Germany) operating at a
frequency of 500 MHz. All 1-D experiments were recorded at five different
temperatures over the range 300-320K. The temperature coefficients (GGG7) of the
amide protons were calculated by analyzing the chemical shifts at these five
temperatures. Two-dimensional experiments were performed at 305 K or 310 K
depending on the quality and clarity of the spectra.
The structure of the peptides is analyzed by using DYANA software (170).
The final DYANA structures were modeled and visualized with the software
package QUANTA using the CHARMM program running on a Silicon Graphics
workstation (171).
27
7. RESULTS
7.1. Identification of PSA-binding peptides (I)
1) PSA-binding peptides
We selected 14 peptide sequences from four different phage display peptide
libraries. The peptides isolated from each library contained characteristic
consensus amino acids in addition to the cysteines engineered in fixed positions
(Table 4).
Table 4. Amino acid sequences of PSA-binding peptides.
Code
A-1*
A-2
Origination
library
CX8C
Peptide sequence
CVFTSDYAFC
CVIYDGNHWC
No. of
isolates
2
2
CI FEPDYSYC
2
A-3
CX8C
CX8C
A-4
CX8C
CVFDDLYSFC
1
B-1
CTFSVDYKYLMC
15
B-2*
CX10C
CX10C
CVFAHNYDYLVC
2
B-3
CX10C
CRFDKEYRTLVC
1
C-1
CX3CX4CX2C
CX3CX4CX2C
CVSYCLFEFCYVC
2
CVEYCWEGSCYVC
7
CVAYCEEWECYVC
1
C-4*
CX3CX4CX2C
CX3CX4CX2C
CVAYCIEHHCWTC
3
C-5
CX3CX4CX2C
CVSYCDGLQCWMC
1
D-1*
CX3CX3CX3C
CX3CX3CX3C
CLSTCAQSCRISC
7
CLLYCHDACWWVC
2
C-2
C-3
D-2
Single-stranded DNA was purified from phage clones after three rounds of selection
with PSA. Peptide sequences were deduced from the DNA sequences of the
corresponding region of the phage genome. Consensus residues found in several clones
are indicated in boldface. Peptides produced as glutathione S-transferase (GST) fusion
proteins are indicated with an asterisk.
28
Four PSA-binding peptides displaying the strongest binding were
constructed as GST fusion proteins. Peptide C-4 with the sequence
CVAYCIEHHCWTC showed the strongest binding to PSA both as a GST-peptide
(Fig. 6) and when expressed on a phage. Two other selected peptides, B-2
(CVFAHNYDYLVC) and A-1 (CVFTSDYAFC), bound to PSA but less efficiently
than C-4. The fourth peptide (D-1) produced as a GST fusion protein did not
show significant binding to PSA even as the corresponding phage clone bound to
PSA.
300000
C-4 (CVAYCIEHHCWTC)
B-2 (CVFAHNYDYLVC)
A-1 (CVFTSDYAFC)
Binding to PSA (CPS)
250000
200000
150000
100000
50000
0
0
25
50
75
100
125
150
175
200
Peptide concentration (nM)
Fig. 6. Binding of GST-peptides to immobilized PSA.
Various amounts of each GST-peptide were added to wells containing PSA captured by
MAb 5E4. The binding of GST-peptides was quantified by IPMA. Data represent mean
values from duplicate wells ± standard errors (SE).
The gel filtration of GST-C-4 revealed four peaks (Table 5, Fig. 7). The
molecular sizes of the peaks were > 500 kDa, 100 kDa, 30 kDa, and about 3 kDa.
All of these except the 3-kDa peak bound to PSA in IPMA (Table 5, Fig. 7). This
indicates that the GST-peptide exists as a 30-kDa monomer, an oligomer (about
100 kDa), and a polymer. The polymeric GST-peptide showed the strongest
binding to PSA, as indicated by the response in relation to the protein absorbance
at 280 nm. The GST fusion protein contains the thrombin recognition sequence
and can be cleaved with thrombin. Thrombin treatment of GST-C-4 caused
29
nearly complete cleavage into 30-kDa (GST only) and 3-kDa components. The 3kDa components consisted of the estimated 31-residue long peptide cleaved from
the GST-peptide by thrombin. The 30-kDa component retained about 5% of the
response in the GST-peptide IFMA in comparison with the noncleaved GST-C-4
(Table 5). In inhibition experiments, the 3-kDa peak reduced the binding of GSTpeptide to PSA by about 30% (Fig. 7).
Table 5. Comparison of the activities of different molecular size forms of GST-C-4.
Fusion protein
GST-C-4
Thrombincleaved
GST-C-4
RMM
(kDa)
~3
~ 30
~ 100
> 500
~3
~ 30
Protein
(% of total)
3.4
83.4
11.4
1.7
17
83
Binding activity
(% of total)
ND
53
26
21
ND
5.2*
In gel filtration, GST-C-4 and thrombin-cleaved GST-C-4 revealed four and two peaks,
respectively. The percentage of protein in each peak was estimated by absorbance at 280
nm. The binding activity was calculated from the IPMA assay.
RMM, relative molecular mass. ND, Cannot be detected. *Denotes binding activity
remaining after thrombin treatment.
2) Properties and specificity of PSA-binding peptides
To determine the binding site of the peptides on PSA, inhibition experiments
were performed with MAbs binding to various epitopes on PSA. Antibodies
binding to epitope regions, which in complexed PSA are covered by the serpin
D1antichymotrypsin (ACT) (also called free-specific antibodies), inhibited the
binding of the peptides most strongly (>80%). MAbs binding to epitopes distant
from the active site of PSA showed a lower degree of inhibition (Table 6).
The binding of GST-peptide fusion proteins (B-2 and C-4) to PSA was blocked
in a dose-dependent fashion by the corresponding synthetic peptide (Table 7).
No significant difference was present in reactivity between peptides with and
without a spacer.
30
When Zn2+, a cation known to bind to PSA, was included in the assay buffer,
the binding activity of peptides was increased. Zn2+ had a dose-dependent effect
and the maximal increase in binding response was at a concentration of 200
µmol/L.
800000
100
C-4
Cleaved C-4
Inhibition (%)
90
700000
669 150
43
1.3
80
600000
500000
60
400000
50
40
300000
Inhibition (%)
Binding to PSA (CPS)
70
30
200000
20
100000
10
0
0
0
10
20
30
40
50
Fraction no.
Fig. 7. Fractionation of the GST-C-4 by gel filtration.
After fractionation of GST-C-4 or thrombin-treated GST-C-4, 5 µL of each fraction was
first incubated in wells containing PSA bound by MAb 5E4. The binding was measured
by IPMA. The arrows show the elution of molecular size standards (669 kDa, 150 kDa,
43 kDa, and 1.3 kDa). The cross symbol (x) shows the inhibition of the binding of GSTpeptide induced by the peptide cleaved from GST-peptide. For this test, 200 µL of each
fraction containing free peptide was first incubated in wells containing PSA captured by
MAb 5E4. After 1 h, the wells were emptied and GST-C-4 (167 nmol/L) was added. After
incubation for 1 h, the binding of GST-peptide was measured by IPMA. Data represent
mean values from duplicate wells.
31
Table 6. Inhibition of GST-peptide binding to PSA by PSA-MAbs.
ISOBM-MAb
Epitope
group
Code (name)
25 (5A10)
26 (9B10)
40 (2E9)
90 (41R5)
57 (H50)
89 (109R75)
86 (PSM773)
Control
1
1
2-a
2-b
3-a
3-b
5
6
GST-peptide
A-1
++
++
+
+
+
+
+
-
B-2
++
++
+
+
+
+
+
-
C-4
++
++
+
+
+
+
+
-
PSA-MAbs representing four epitope groups on PSA were used to compete with the
GST-peptides for binding to PSA. An antibody from group 6, in which MAb 5E4
belongs, was used as a negative control. Each MAb (33.3 nM) was incubated with PSA
captured by 5E4 coated onto microtitration wells. After washing, GST-peptide was
added. The binding of GST-peptides was quantified by fluorometry.
Reduction of binding of GST-peptide to solid-phase PSA (%)
++ = >
80%
+ =
60-80%
- =
No inhibition
Table 7. Effect of synthetic peptides with and without a spacer on the binding of
GST-peptides to immobilized PSA.
Inhibition by synthetic peptide (%)
Concentration
1.7 µM
17 µM
34 µM
B-2
39
71
75
B-2 linker
40
72
78
C-4
41
81
90
C-4 linker
31
70
80
Various amounts of synthetic peptides and the corresponding GST-peptide (0.17 µM)
were added to wells containing PSA bound by MAb 5E4. The binding of GST-peptides
was quantified by fluorometry.
32
When analyzed by isoelectric focusing, the main form of peptide-bound PSA
was PSA-B (pI 6.9), while PSA-A (pI 7.2) formed only a faint band (Fig. 4 in the
original publication II). Unbound PSA also displayed a major band at pI 6.9
(corresponding to PSA-B, -C and -D) and a faint band at pI 6.7 (PSA-E) (Fig. 4 in
the original publication II). When PSA isoenzymes A, B, C, D, and E purified by
anion exchange chromatography were fractionated on the peptide column, about
70% of “intact” isoenzymes A and B bound to the column, as compared with 25%
of PSA C, 12% of PSA-D, and 7% of PSA-E.
0.30
33.3 µM
3.3 µM
Absorbance 405 nm
0.25
0.20
0.33 µM
0.15
0.17 µM
0.10
0 µM
0.05
0.00
0
20
40
60
80
Time (min)
Fig. 10. Effect of GST-C-4 on the enzymatic activity of PSA.
PSA (0.33 µM) was incubated with increasing concentrations of GST-C-4 (0 to 100-fold
molar excess) for 1 h, after which the chromogenic substrate S-2586 was added and
enzymatic activity was monitored by measuring the absorbance at 405 nm. Data
represent mean values from duplicate wells ± SE.
The enzymatic activity of PSA separated with the peptide column was
analyzed by reacting bound and unbound forms with ACT. After 24 h, about 77%
of peptide-bound PSA and 30% of unbound PSA had formed a complex. The
reactivity of peptide-bound PSA was similar to that of PSA isoenzyme B, and that
of unbound PSA was similar to that of PSA isoenzyme C.
36
between residues Ala-4 and Tyr-7 for B-2, and a type II E-turn between Ile-6 and
His-9 for C-4. However, the most important stabilizing effect for the peptide
structures was the disulfide bridge between the N- and C-terminal cysteines,
peptide C-4 having an additional disulfide bond between Cys-5 and Cys-10,
enabling a tight turn in the sequence between these amino acids.
The effect of peptides and alanine replacement derivatives on the enzymatic
activity of PSA, showed that most of the changes caused inactivation or
reduction of the enzyme activating capacity on PSA. However, replacement of
His-5 by Ala-5 in B-2 increased the activity. In peptide C-4, the effect was
strongly decreased when Tyr-4, Ile-6, and Try-11 were replaced by alanine.
Analysis of the binding specificity of peptides on PSA by a cross-inhibition
experiment showed that binding of GST fusion protein C-4 to PSA was blocked
by each of the synthetic peptides A-1, B-2, and C-4 in a dose-dependent fashion
(Fig. 4 in the original publication IV). At a 100-fold molar excess of the synthetic
peptides, the inhibition was between 60% and 70%. This suggests that the
peptides bind to the same site on PSA.
39
8. DISCUSSION
8.1. Identification and characteristics of novel PSA-binding ligands
PSA is a serine protease and a sensitive serum marker for PCa. Measurement of
serum PSA is widely used for early detection and management of PCa patients.
However, false-positive results caused by BPH limit the utility of the PSA assay
for diagnosis of PCa. Measurement of the proportions of free PSA and the PSAACT complex has been used to reduce the number of false-positive results by 2030% (172, 173). However, even with this approach, the frequency of falsepositive results is over 50%. Recent studies suggest that detection of variants of
free PSA could improve discrimination between PCa and BPH (14, 15). We
therefore aimed at identifying specific ligands for cancer-related forms of PSA.
Phage libraries can display 109 different peptides on the surface of phage. The
peptides are expressed as a fusion with phage surface proteins. We initially
coated PSA directly onto microtitration wells, but with this method no PSAbinding phage could be isolated. Apparently, direct immobilization of PSA onto
the wall of the microtitration well ether changes the PSA structure or causes it to
adopt an unfavorable orientation for biopanning. We changed the strategy by
using a MAb, which does not block the active site, to capture PSA. With this
approach, 14 PSA-binding peptides were identified. We have screened several
linear phage display peptide libraries, but no peptides were isolated. From each
cyclic phage peptide library, by contrast, several sequences could be identified.
This strongly suggests that the essential structure of the peptide-libraries is
important and a cyclic conformation is necessary for binding and is more stable
for the interaction with PSA. Among the selected peptides, bicyclic peptides
(with four cysteines) showed the highest affinity. Phage fUSE5 expresses 3-5
copies of the pIII protein fused to peptide inserts containing the same sequence,
resulting in multivalent binding (174).
To facilitate studies on the binding properties and specificity of the peptides,
they were produced as GST-peptide fusion proteins. These fusion proteins
contained three molecular size forms: monomer, oligomer and polymer. The
polymer displayed the strongest binding to PSA, followed by the oligomer and
the monomer, this indicates that multivalent binding enhances binding avidity.
In the presence of Zn2+, the affinity constants of the GST-peptides increased 3to 7-fold, suggesting involvement of Zn2+ in the binding between PSA and
peptides. Surface plasmon resonance (BIA core) experiments showed that the
effect of Zn2+ was explained by a decrease in dissociation rate rather than an
increase in association rate. This may be mediated by Zn2+ chelated between
amino acid residues of PSA and the peptide or by altering the structure of PSA.
40
Interestingly, zinc inhibits the enzymatic activity of PSA (17, 175). Another
possibility is that Zn2+ stabilizes the 3-D structure of the peptide in a way similar
to the interaction of Zn2+ with DNA-binding zinc finger proteins (176, 177).
The peptides did not bind to chymotrypsin and cathepsin G, which have an
enzymatic specificity similar to that of PSA. Neither did they bind to hK2, which
is structurally closely related to PSA and shows 79% identity at the amino acid
level (76). In addition, The GST-peptides did not bind to PSA-serpin complexes
occurring in serum, i.e. PSA-ACT and PSA-API, in which the serpins cover the
peptide-binding region on PSA. The binding of peptides to PSA was also blocked
by antibodies that react with free PSA through epitopes covered in PSA-serpin
complexes; moreover, these MAbs inhibit the enzymatic activity of PSA (168).
Thus, the peptides are specific to enzymatically active free PSA. The peptides
enhanced the enzymatic activity of PSA against the synthetic peptide substrate S2586, and the effect correlated with the affinity of the peptides to PSA. These
results suggest that the peptides bind to the vicinity of the active site, changing
the conformation of PSA, which possibly makes the catalytic pocket more
accessible to synthetic substrates.
8.2. Conformational and biochemical analyses of PSA-binding
peptides
PSA-binding peptides (A-1, B-2, and C-4) contain the aromatic amino acid
tyrosine. The other consensus amino acids, phenylalanine, valine, and leucine,
have hydrophobic side-chains. Replacement of the key amino acid tyrosine with
alanine caused total loss of binding to PSA. Replacement of alanine for other
consensus amino acids also reduced the binding activity. These findings suggest
that amino acids containing aromatic or hydrophobic side-chains are the contact
residues with PSA, and they are responsible for the binding and spatial
arrangement between the peptide and PSA. Replacement of nonconsensus amino
acids with alanine did not cause loss of binding to PSA. Thus, other amino acids,
as elements of the peptide backbone, play a role in the conformation of peptides
but are not essential for binding.
NMR analysis suggested that peptide cyclization was induced in turn
conformations, which are essential for the effect on the enzymatic activity of
PSA. Apparently, the turn structures of the peptides place the binding residues
in an exposed conformation for interaction with PSA.
41
8.3. Separation of enzymatically active and inactive PSA by peptide
affinity chromatography
The peptides were verified by synthesizing them chemically and preparing an
affinity column linking the peptide through a spacer to the matrix. The spacer
does not affect the function of the peptides but preserves their activity after
immobilization. When PSA comprising various forms was fractionated by
peptide affinity chromatography, the intact, enzymatically active PSA was
retained and could be isolated. ProPSA, which is enzymatically inactive, did not
bind to the peptide, but after activation by trypsin about 56% bound to the
peptide column. When serum samples from PCa patients were applied to the
column, a minor fraction of free PSA bound to the column, suggesting that some
enzymatically active PSA remains free in circulation. These studies demonstrate
that the PSA-binding peptides have unique specificity and recognize
enzymatically active free PSA.
8.4. Immunopeptidometric assay for enzymatically active PSA
Reagents specifically recognizing the active form of PSA are potentially useful for
PCa diagnostics, but antibodies specific to active PSA are not available.
Antibodies bind to epitopes on the surface of PSA (168) and do not recognize
structural differences within grooves and pits of the protein. Short cyclic
peptides containing 10-13 amino acids apparently have the ability to recognize
differences in the inner structure of PSA.
In PCa, the normal structure of the prostate is deranged by disruption of the
basal cell layer and basement membrane. PSA may therefore directly be released
into the extracellular space and reach the circulation. This could explain the high
proportion of PSA complexed with inhibitors in PCa patients (61). When
enzymatically active PSA purified from seminal fluid is added to serum, most of
it binds to A2M and ACT, but a small portion remains free. Our finding of active
PSA in patient samples indicates that the reaction between PSA and inhibitors
leaves part of the active PSA free.
The IPMA is a new assay principle based on the use of peptides as a tracer in
combination with a monoclonal capture antibody. All forms of PSA were
captured by the immobilized MAb, but only enzymatically active free PSA was
recognized by the peptide. By using an immunofluorescent enzymatic assay,
Niemelä et al. found that about 3% enzymatically active free PSA occurs in serum
of PCa patients (13). Our finding is in line with this result. The IPMA, thus, offers
a potential approach for detection of cancer-associated forms of PSA. However,
the sensitivity of the IPMA needs to be improved in order to detect active PSA at
clinically important concentrations, i.e. 3-10 µg/L.
42
9. SUMMARY
PSA, as the most important marker, is widely being used for diagnosis and
monitoring of PCa, but the high frequency of false-positive results is problematic.
The purpose of this study was to identify novel PSA-binding peptides that can be
utilized as reagents for development of more cancer-specific diagnostic methods
and as tools for targeting PCa.
First, a new strategy for selection of phage-displayed peptides was
developed. By using a monoclonal PSA antibody, which does not block the active
site of the enzyme, PSA was immobilized on microtitration wells. Fourteen PSAbinding peptides were selected from four cyclic phage libraries. To facilitate
studies on the binding properties and specificity of the peptides, representative
peptides from each phage library were produced as GST fusion proteins and also
synthesized chemically. Bicyclic peptide C-4 (with four cysteines) showed the
highest binding affinity to PSA. The peptides did not bind to other serine
proteinases or to PSA-serpin complexes occurring in serum. The peptides
enhanced the enzymatic activity of PSA. These results suggest that the peptides
bind to the vicinity of the active site, changing the conformation of PSA.
The conformation of PSA-binding peptides was analyzed by NMR, and the
effect of alanine replacement derivatives on the enzymatic activity of PSA was
studied. The results suggest that the effect of the peptides on PSA enzymatic
activity is based on the interaction with aromatic and hydrophobic side-chains of
amino acids in the PSA-binding peptides. The cysteine disulfide bond of the
peptides plays an important role in stabilizing the peptide structure.
A peptide affinity column was prepared by linking the peptide through a
spacer to the Sepharose gel. Intact, enzymatically active free PSA was bound to
the peptide affinity column, whereas internally cleaved forms and proPSA were
not. When serum from PCa patients was applied to the peptide column, a minor
fraction of free PSA could be separated from other forms of PSA.
An immunopeptidometric assay (IPMA) for enzymatically active PSA was
developed. All forms of PSA were captured by a PSA-MAb onto the solid phase,
and a peptide fusion protein to PSA was used as a tracer to quantitate the
enzymatically active PSA. The IPMA detected enzymatically active PSA purified
from seminal fluid and proPSA from LNCaP cell culture medium after activation
by trypsin. The minor fraction of free PSA in serum of PCa patients was
recognized by the IPMA. Thus, this new assay principle has wide potential
utility.
In conclusion, PSA-binding peptides with unique specificity were identified
by phage display. These peptides, containing 10-13 amino acids, have the ability
to recognize the inner structure of PSA and may be able to diffuse into tissues
43
and target PSA-secreting cells. These peptides are thus promising tools for the
development of novel diagnostic methods and targeted therapeutics for PCa.
44
10. ACKNOWLEDGMENTS
This study was carried out at the Department of Clinical Chemistry of the
University of Helsinki. I thank Professor Ulf-Håkan Stenman, head of the
department, for providing excellent working facilities.
Professor Ulf-Håkan Stenman was also the supervisor of this thesis and had
introduced me to cancer research field. I am most grateful for his guidance and
support throughout the study. I deeply admire his vast scientific knowledge and
his ability to solve the various problems encountered.
Docent Pirjo Laakkonen and Professor Kim Pettersson, the official reviewers
of this thesis, are thanked for their careful and efficient review and constructive
criticism and comments.
I am grateful for having had the opportunity to work with Docent Erkki
Koivunen, Jari Leinonen, PhD, Docent Ale Närvänen, Docent Hilkka Lankinen,
Miikka Pakkala, MSc, and Lei Zhu, MSc. Their collaboration and advice were
valuable. I also thank my other coauthors Professor Jouko Vepsäläinen, Anu
Jylhäsalmi, MSc, Harri Santa, PhD, and Mikael Peräkylä, PhD for their
contribution to this work.
Docents Ursula Turpeinen, Riitta Koistinen, Outi Monni, Heli Nevanlinna,
and Ralf Bützow are gratefully acknowledged for their advice, and contributing
to the wonderful atmosphere in the lab.
I warmly thank Helena Taskinen, Taina Grönholm, Anne Ahmanheimo,
Maarit Leinimaa, Marianne Niemelä, Kätli Santila, Liisa Airas, Arja Mikkola,
Liisa Kurkela, Rauni Lehtola, Inkeri Kasso and Sanna Kihlberg for technical
assistance, sample collection and friendship throughout the years.
I am indebted to my colleagues and friends Patrik Finne, Annukka Paju, Oso
Rissanen, Jakob Stenman, Susanna Lintula, Kristina Hotakainen, Erik Mandelin,
Hannu Koistinen, Henrik Alfthan, and Leena Valmu for valuable discussions
about research work and different help in many practical aspects.
I thank my colleagues and friends Pia Vahteristo, Piia Vuorela, Laura
Sarantaus, Johanna Tapper, Sirpa Stick, Laura Salonen, Anitta Tamminen, Jan
Andersén, Henrik Edgren, Siina Junnila, Can Hekim, Anna Järvinen, Sami
Kilpinen, Maija Wolf, Tuula Airaksinen, Henna Nieminen, Gynel Arifdsan,
Annikki Löfhjelm, Heini Lassus, Sari Tuupanen, Reetta Jalkanen, and all others
in the research laboratory. You have all contributed to the good working
atmosphere.
Carol Ann Pelli, HonBSc, is acknowledged for her skillful revision of the
English language of this thesis.
I am grateful for the financial support from the Finnish National Technology
Agency (TEKES), the Research Foundation of Helsinki University Central
45
Hospital (EVO), University of Helsinki, the Sigrid Juselius Foundation, the
Finnish Academy of Sciences, and the Finnish Cancer Foundation.
Many thanks to all of my friends in and outside of Finland for their assistance
with all manner of things. I particularly thank my friends Hanneli, Pekka,
Anneli, and Eero for their help whenever I met with difficulties.
I am deeply grateful to my parents for their everlasting love, care, and
support, to my sister for her unquestioning loyalty and care, and to my son Junjie
for his understanding and support with whatever problems had to be overcome.
Helsinki, October 2004
46
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