Simple purification procedure for human prostatic kallikrein hK2 in its active form

Kallikrein hK2 is a new potential marker of prostate cancer. It is the last member of the human kallikrein gene family to be isolated. We propose a simple purification procedure permitting us to obtain the active form of hK2 starting from human seminal plasma and using commonly available chromatography matrices. In contrast to recently published papers, this procedure is carried out without any immunoaffinity chromatography step and without the need for any antibody to follow the purification. Furthermore, it does not require any recombinant DNA technology nor sophisticated instruments.     

Large-scale purification of a stable form of recombinant tobacco etch virus protease

Tobacco etch virus NIa proteinase (NIa-Pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro. We have designed a mutant NIa-Pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pure, active, and stable over time. Histidine-tagged forms of both wild-type and mutant NIa-Pro were overexpressed in E. coli under conditions in which greater than 95% of the protease was in the insoluble fraction after cell lysis. An inclusion body preparation followed by denaturing purification over a single affinity column and protein renaturation yields greater than 12.5 mg enzyme per liter of bacterial cell culture. NIa-Pro purified according to this protocol has been used for quantitative removal of fusion domains from a variety of proteins prepared for crystallization and biochemical analysis.     

Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors.

Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 x 10(5) M-1 x s-1) and the high concentrations of PCI (4 microM) and hK2 (0.2 microM) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ at micromolar concentrations well below the 9 mM zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control.     

Characterization of the gene for prostate-specific antigen, a human glandular kallikrein

The gene for the human glandular kallikrein, prostate-specific antigen, has been cloned. The sequence of 7130 nucleotides encompassing the gene and 633 bp of 5' and 639 bp of 3' flanking DNA has been determined. The translation initiation site was slightly heterogeneous, yielding 5' non-translated leader sequences of 41 and 35 bp. The gene is divided into five exons, with introns located at positions identical with those found in other glandular kallikrein genes. The nucleotide sequence is very similar to that of the human kallikrein gene hGK-1, with 76 to 93% of the nucleotides being identical in the exons and 76 to 87% in the introns. The similarity also extends approximately 200 bp into the sequence flanking the 5' end of hGK-1 and several other, both human and rodent, glandular kallikrein genes.     

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.     

Determination and analysis of antigenic epitopes of prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2) using synthetic peptides and computer modeling.

Prostate specific antigen (PSA) and human glandular kallikrein 2 (hK2), produced essentially by the prostate gland, are 237-amino acid monomeric proteins, with 79% identity in primary structure. Twenty-five anti-PSA monoclonal antibodies (Mabs) were studied for binding to a large array of synthetic linear peptides selected from computer models of PSA and hK2, as well as to biotinylated peptides covering the entire PSA sequence. Sixteen of the Mabs were bound to linear peptides forming four independent binding regions (I-IV). Binding region I was localized to amino acid residues 1-13 (identical sequence for PSA and hK2), II (a and b) was localized to residues 53-64, III (a and b) was localized to residues 80-91 (= kallikrein loop), and IV was localized to residues 151-164. Mabs binding to regions I and IIa were reactive with free PSA, PSA-ACT complex, and with hK2; Mabs binding to regions IIb, IIIa, and IV were reactive with free PSA and PSA-ACT complex, but unreactive with hK2; Mabs binding to region IIIb detected free PSA only. All Mabs tested (n = 7) specific for free PSA reacted with kallikrein loop (binding region IIIb). The presence of Mabs interacting with binding region I did not inhibit the catalytic activity of PSA, whereas Mabs interacting with other binding regions inhibited the catalysis. Theoretical model structures of PSA, hK2, and the PSA-ACT complex were combined with the presented data to suggest an overall orientation of PSA with regard to ACT.     

Large-scale purification of functional recombinant human aquaporin-2

The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT-AQP2 could be obtained per liter of bioreactor culture. HT-AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93+/-0.03x10(-13) cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large-scale expression of functional recombinant human AQP2 that is suitable for structural studies.     

Purification and characterization of a high-molecular-weight form of recombinant human Interleukin-2

During purification of recombinant Interleukin-2 (rIL-2) by reversed-phase HPLC, early fractions are discarded due to the presence of an unidentified form of rIL-2. A procedure has been developed to isolate and purify this unidentified form of rIL-2. The purification process involves two chromatography steps and utilizes a Bakerbond Carboxy-Sulfon (CS) column under two different conditions. This material, designated as a high-molecular-weight form of rIL-2 (HMWrIL-2), exhibits lower mobility during SDS-PAGE and has apI which is approximately one unit less than that of rIL-2, but has similar bioactivity to rIL-2. Structural analysis through enzymatic cleavage, HPLC peptide mapping, mass spectrometry, sequencing, and amino acid composition revealed that the difference between these two proteins is a C-terminal extension of 11 amino acids. This extension could be the result of a nonstandard translation event.     

Evidence that cervical cancer cells secrete IL-2, which becomes an autocrine growth factor

We present evidence that cervical cancer cells express a functional IL-2 receptor (IL-2R). In fact, by RT-PCR we obtained that the IL-2R is present in CALO, and INBL cells, and that it consisted of the αIL-2R, βIL-2R, and γIL-2R chains. We also found that IL-2 is a growth factor for these cell lines, and unexpectedly that CALO and INBL themselves being cancer cells produce, and secrete IL-2. Antibodies against the α and β subunits of the IL-2R inhibited cell proliferation thus hinting to a cell growth dependency on this factor. Our results thus provide evidence that the IL-2R on cervical cancer cells is part of an autocrine mechanism for its growth to the extent that, like lymphocytes, they produce and become partially dependent on this growth factor. We think that in view of our results caution should be taken when IL-2 is being considered for cancer therapy; in particular when the patient’s cancer cells present the IL-2R, because as indicated by our results, the use of this factor could promote tumor growth. Finally, the possible implications of the expression of both IL-2, and IL-2R on cervical cancer cells on the immune escape mechanism of tumor cells are discussed.     

Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates.

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747-2754]. Selected substrates were then transplanted into the reactive site loop of alpha1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific alpha1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.     

High level of expression in the prostate of a human glandular kallikrein mRNA related to prostate-specific antigen

Using a synthetic oligonucleotide primer complementary to human prostate-specific antigen mRNA, we found that an additional sequence possibly similar to human glandular kallikrein-1 could be read by a primer-extension sequencing technique. We were able to confirm the identity of that additional sequence with another oligonucleotide primer complementary to a specific region of the human glandular kallikrein-1 mRNA sequence. Northern blot analysis with 2 oligonucleotide probes respectively specific for prostate-specific antigen and human glandular kallikrein-1 mRNAs showed that the length of both mRNAs was similar at 1.5 kb. The level of human glandular kallikrein-1 mRNA relative to that of prostate-specific antigen could be estimated as approx. 10-20%. This study constitutes the first evidence that the human glandular kallikrein-1 gene is expressed at a high level in a human tissue.     

Biochemical and enzymatic characterization of human kallikrein 5 (hK5), a novel serine protease potentially involved in cancer progression

Human kallikrein 5 (KLK5) is a member of the human kallikrein gene family of serine proteases. Preliminary results indicate that the protein, hK5, may be a potential serological marker for breast and ovarian cancer. Other studies implicate hK5 with skin desquamation and skin diseases. To gain further insights on hK5 physiological functions, we studied its substrate specificity, the regulation of its activity by various inhibitors, and identified candidate physiological substrates. After producing and purifying recombinant hK5 in yeast, we determined the k(cat)/K(m) ratio of the fluorogenic substrates Gly-Pro-Arg-AMC and Gly-Pro-Lys-AMC, and showed that it has trypsin-like activity with strong preference for Arg over Lys in the P1 position. The serpins alpha(2)-antiplasmin and antithrombin were able to inhibit hK5 with an inhibition constant (k(+2)/K(i)) of 1.0 x 10(-) (2)and 4.2 x 10(-4) m(-1) min(-1), respectively. No inhibition was observed with the serpins alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, although alpha(2)-macroglobulin partially inhibited hK5 at high concentrations. We also demonstrated that hK5 can efficiently digest the extracellular matrix components, collagens type I, II, III, and IV, fibronectin, and laminin. Furthermore, our results suggest that hK5 can potentially release (a) angiostatin 4.5 from plasminogen, (b) "cystatin-like domain 3" from low molecular weight kininogen, and (c) fibrinopeptide B and peptide beta15-42 from the Bbeta chain of fibrinogen. hK5 could also play a role in the regulation of the binding of plasminogen activator inhibitor 1 to vitronectin. Our findings suggest that hK5 may be implicated in tumor progression, particularly in invasion and angiogenesis, and may represent a novel therapeutic target.     

Endothelial cells secrete a factor that promotes fibroblast contraction of hydrated collagen gels

Bovine aortic endothelial cells (BAEC), grown in vitro, are shown to synthesize and secrete factor(s) that stimulate fibroblasts to contract collagen matrices. The amount of contraction-promoting activity in the conditioned media is dependent on conditioning time and the number of cells in the culture. Production of the contraction-promoting activity continues at a high stable level for at least 5 d in serum-free medium but is abolished when the cells are exposed to an inhibitor of protein synthesis. The mechanism of action of the contraction factor(s) derived from endothelial cells was compared with that of unidentified serum factors. The endothelial cell-secreted factor(s) depends on active protein synthesis by the target cell but does not need to be present during the contraction process. The serum factors on the other hand promote collagen contraction in the absence of de novo protein synthesis but need to be continuously present. Preliminary biochemical characterization of the contraction-promoting factors produced by endothelial cells revealed properties similar to those of previously identified growth factors. However, the BAEC-secreted factor was found to be distinct from a previously identified contraction-promoting transforming growth factor beta.     

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker.     

High-frequency transfection of cultured human epidermal basal cells that differentiate to form a multilayered tissue

Primary cultures of human keratinocytes form a multilayered tissue. By incubating the tissue cultures in Ca2(+)-free medium the differentiated cell layers can be stripped off leaving a basal cell monolayer. We have developed a method for high-frequency transfection of these epidermal basal cells with genes inserted into Epstein-Barr virus-based expression vectors. Using the Escherichia coli lac z gene as a marker gene, the transient and long-term expression and the fate of the transfected cells were studied. During regeneration of the multilayered tissue most of the transfected basal cells enlarge and undergo differentiation, but a minor population remains as basal cells. Incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate results in an increase in the proportion of transfected keratinocytes that are small, suggesting a relative expansion of the immature cell pool.     

Expression, purification, and characterization of stable, recombinant human adenylosuccinate lyase

The full length human adenylosuccinate lyase gene was generated by a PCR method using a plasmid encoding a truncated human enzyme as template, and was cloned into a pET-14b vector. Human adenylosuccinate lyase was overexpressed in Escherichia coli Rosetta 2(DE3)pLysS as an N-terminal histidine-tagged protein and was purified to homogeneity by a nickel-nitriloacetic acid column at room temperature. The histidine tag was removed from the human enzyme by thrombin digestion and the adenylosuccinate lyase was purified by Sephadex G-100 gel filtration. The histidine-tagged and non-tagged adenylosuccinate lyases exhibit similar values of Vmax and Km for S-AMP. Analytical ultracentrifugation and circular dichroism revealed, respectively, that the histidine-tagged enzyme is in tetrameric form with a molecular weight of 220 kDa and contains predominantly alpha-helical structure. This is the first purification procedure to yield a stable form of human adenylosuccinate lyase. The enzyme is stable for at least 5 days at 25 degrees C, and upon rapid freezing and thawing. Temperature as well as reducing agent (DTT) play critical roles in determining the stability of the human adenylosuccinate lyase.     

Properties of a cleaved two-chain form of recombinant human growth hormone

Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.     

Production of a biologically active variant form of recombinant human secretin

A biologically active variant form of recombinant human secretin was produced using a gene fusion system designed to facilitate the purification of the protein. The fusion protein was recovered from the culture medium of Escherichia coli by IgG affinity chromatography, and recombinant secretin was released by cyanogen bromide treatment. A novel approach involving addition of a C-terminal Gly-Lys-Arg extension, was used to overcome the lack of amidation of recombinant proteins in Escherichia coli. The biological activity of the recombinant variant of secretin was at least 80% of the porcine secretin standard.