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Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. Timely administration of antibiotics approved for the treatment of anthrax disease may prevent associated morbidity and mortality. However, any delay in initiating antimicrobial therapy may result in increased mortality, as inhalational anthrax progresses rapidly to the toxemic phase of disease. An anthrax antitoxin, AVP-21D9, also known as Thravixa (fully human anthrax monoclonal antibody), is being developed as a therapeutic agent against anthrax toxemia. The efficacy of AVP-21D9 in B. anthracis-infected New Zealand White rabbits and in cynomolgus macaques was evaluated, and its safety and pharmacokinetics were assessed in healthy human volunteers. The estimated mean elimination half-life values of AVP-21D9 in surviving anthrax-challenged rabbits and nonhuman primates (NHPs) ranged from approximately 2 to 4 days and 6 to 11 days, respectively. In healthy humans, the mean elimination half-life was in the range of 20 to 27 days. Dose proportionality was observed for the maximum serum concentration (Cmax) of AVP-21D9 and the area under the concentration-time curve (AUC). In therapeutic efficacy animal models, treatment with AVP-21D9 resulted in survival of up to 92% of the rabbits and up to 67% of the macaques. Single infusions of AVP-21D9 were well tolerated in healthy adult volunteers across all doses evaluated, and no serious adverse events were reported. (This study has been registered at ClinicalTrials.gov under registration no. NCT01202695.)., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Development of anthrax countermeasures that may be used concomitantly in a postexposure setting requires an understanding of the interaction between these products. Anthrax immune globulin intravenous (AIGIV) is a candidate immunotherapeutic that contains neutralizing antibodies against protective antigen (PA), a component of anthrax toxins. We evaluated the interaction between AIGIV and BioThrax (anthrax vaccine adsorbed) in rabbits. While pharmacokinetics of AIGIV were not altered by vaccination, the vaccine-induced immune response was abrogated in AIGIV-treated animals.

Ku is a heterodimer composed of p70 and p80, and is the regulatory subunit of DNA-dependent protein kinase. As a multifunctional DNA-binding protein complex, Ku plays important roles in DNA damage repair through non-homologous end joining and in V(D)J recombination. In addition, Ku has also been implicated in various biological functions including growth control, cell proliferation, cell cycle, chromosome maintenance, transcriptional regulation, apoptosis, and viral infection. In particular, using our Inverse Genomics (Immusol, Inc., San Diego, CA) platform technology, we recently identified Ku80 as an essential co-factor for human immunodeficiency virus replication. Although Ku has been studied extensively in the past years, its in-depth study as well as development as a drug target has been limited by conventional DNA-binding activity assay. Here we describe the development and applications of a nonradioactive DNA binding assay in the 96-well format. We show that this plate-formatted assay is more sensitive and allows for direct quantification when compared with an electrophoretic mobility shift assay. The establishment of this assay will not only facilitate structure and function studies on Ku, but also help the development of Ku protein or its DNA repair enzyme complex as a drug target.

We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M(+)HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M(+)HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M(+)HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.

Many serine proteases play important regulatory roles in complex biological systems, but only a few have been linked directly with capillary morphogenesis and angiogenesis. Here we provide evidence that serine protease activities, independent of the plasminogen activation cascade, are required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro. A homology cloning approach targeting conserved motifs present in all serine proteases, was used to identify candidate serine proteases involved in these processes, and revealed 5 genes (acrosin, testisin, neurosin, PSP and neurotrypsin), none of which had been associated previously with expression in endothelial cells. A subsequent gene-specific RT-PCR screen for 22 serine proteases confirmed expression of these 5 genes and identified 7 additional serine protease genes expressed by human endothelial cells, urokinase-type plasminogen activator, protein C, TMPRSS2, hepsin, matriptase/MT-SP1, dipeptidylpeptidase IV, and seprase. Differences in serine protease gene expression between microvascular and human umbilical vein endothelial cells (HUVECs) were identified and several serine protease genes were found to be regulated by the nature of the substratum, ie. artificial basement membrane or fibrillar type I collagen. mRNA transcripts of several serine protease genes were associated with blood vessels in vivo by in situ hybridization of human tissue specimens. These data suggest a potential role for serine proteases, not previously associated with endothelium, in vascular function and angiogenesis.

Mammalian urokinase-type plasminogen activator (uPA) is produced as a stable single polypeptide chain zymogen and requires a distinct proteolytic cleavage to become an active, two-chain enzyme. In contrast, chicken uPA, both native and recombinant, is found predominantly as a two-chain, active enzyme even in the absence of plasmin, a physiological activator. Here we show that the proclivity to autoactivate is not a unique property of the chicken uPA catalytic domain but requires sequences distinct from and independent of the serine protease domain. Human/chicken chimeric uPA molecules and point mutants were used to determine the structural requirements for uPA autoactivation versus zymogen stability. The amino terminal fragment of chicken uPA engineered onto the human uPA molecule can induce the autoactivation of the human uPA. In fact, the first twenty residues of the chicken uPA are necessary and sufficient to induce the autoactivation of chicken and human uPA. These results indicate that sequence motifs, distal to the active site, control the substrate specificity and catalytic efficiency of uPA activity in autolytic activation.

A quantitative assessment of rate-limiting steps in metastasis has always been challenging because of the difficulty of detecting small tumor cell populations. We have developed a highly sensitive assay for monitoring the metastatic dissemination of human tumor cells in the chick embryo and used this assay to investigate the relative efficacy of sequential stages in the metastatic cascade for two malignant human tumor cells lines, HEp3 and HT1080. This assay is based on the real-time PCR amplification of human alu sequences and exhibits a high sensitivity (25 cells/lung) with a large linear range (50-100,000 cell/lung). The assay is optimized for a high number of replicate in vivo assays (50-100 animals/assay) and can be applied in both experimental and spontaneous metastasis models. Using quantitative alu PCR, we determined that HEp3 spontaneously metastasizes very efficiently and rapidly, generating secondary growth in the lung exceeding 1-2 x 10(4) cells/lung in 7 days. In contrast, spontaneous HT1080 metastasis is 50-100-fold less efficient, resulting in only 200-400 cells/lung in 7 days. By taking advantage of the sensitivity and specificity of the real-time alu PCR assay we were also able to quantitatively assess multiple steps in metastasis including intravasation, arrest of tumor cells in secondary organs of the embryo, and the initial growth and expansion of the arrested tumor cells. A comparative analysis of HEp3 and HT1080 metastasis demonstrates that the relatively low-to-moderate metastatic rate of HT1080 is caused by two distinct deficiencies, an 8-10-fold lower rate of intravasation and a delayed onset of HT1080 growth expansion in the secondary organ. Thus, a very facile metastasis model system coupled with the sensitive, real-time PCR-based assay allows for the identification and quantification of rate-limiting steps in the metastatic cascade for select human tumor cell lines.

The contribution of specific type I collagen remodeling in angiogenesis was studied in vivo using a quantitative chick embryo assay that measures new blood vessel growth into well-defined fibrillar collagen implants. In response to a combination of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), a strong angiogenic response was observed, coincident with invasion into the collagen implants of activated fibroblasts, monocytes, heterophils, and endothelial cells. The angiogenic effect was highly dependent on matrix metalloproteinase (MMP) activity, because new vessel growth was inhibited by both a synthetic MMP inhibitor, BB3103, and a natural MMP inhibitor, TIMP-1. Multiple MMPs were detected in the angiogenic tissue including MMP-2, MMP-13, MMP-16, and a recently cloned MMP-9-like gelatinase. Using this assay system, wild-type collagen was compared to a unique collagenase-resistant collagen (r/r), with regard to the ability of the respective collagen implants to support cell invasion and angiogenesis. It was found that collagenase-resistant collagen constitutes a defective substratum for angiogenesis. In implants made with r/r collagen there was a substantial reduction in the number of endothelial cells and newly formed vessels. The presence of the r/r collagen, however, did not reduce the entry into the implants of other cell types, that is, activated fibroblasts and leukocytes. These results indicate that fibrillar collagen cleavage at collagenase-specific sites is a rate-limiting event in growth factor-stimulated angiogenesis in vivo.

We have isolated a novel 75-kDa gelatinase from a chicken macrophage cell line, HD11. Biochemical and immunological characterization of the purified enzyme demonstrated that it is distinct from the chicken 72-kDa gelatinase A (MMP-2). The enzyme is capable of specific gelatin binding and rapid gelatin cleavage. Incubation with an organomercurial compound (p-aminophenylmercuric acetate) induces proteolytic processing and activation of this enzyme, and the resultant gelatinolytic activity is sensitive to both zinc chelators and tissue inhibitors of metalloproteinases. A full-length cDNA for the enzyme has been cloned, and sequence analysis demonstrated that the enzyme possesses the characteristic multidomain structure of an MMP gelatinase including a cysteine switch prodomain, three fibronectin type II repeats, a catalytic zinc binding region, and a hemopexin-like domain. The 75-kDa gelatinase is produced by phorbol ester-treated chicken bone marrow cells, monocytes, and polymorphonuclear leukocytes, cell types that charac- teristically produce the 92-kDa mammalian gelatinase B (MMP-9). The absence of a 90-110-kDa gelatinase in these cell types indicates that the 75-kDa gelatinase is likely the avian counterpart of gelatinase B. However, the protein is only 59% identical to human gelatinase B, whereas all previously cloned chicken MMP homologues are 75-90% identical to their human counterparts. In addition, the new 75-kDa chicken gelatinase lacks the type V collagen domain that is found in all mammalian gelatinase Bs. Furthermore, the secreted enzyme appears structurally distinct from known gelatinase Bs and the activated enzyme can cleave fibronectin, which is not a substrate for mammalian gelatinase B. Thus the results of this study indicate that a second MMP gelatinase exists in chickens, and although it is MMP-9/gelatinase B-like in its overall domain structure and expression pattern, it appears to be biochemically divergent from mammalian gelatinase B.

The glycine-rich loop, one of the most important motifs in the conserved protein kinase catalytic core, embraces the entire nucleotide, is very mobile, and is exquisitely sensitive to what occupies the active site cleft. Of the three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein kinase (cAPK)], Gly(52) is the most important for catalysis because it allows the backbone amide of Ser(53) at the tip of the loop to hydrogen bond to the gamma-phosphate of ATP [Grant, B. D. et al. (1998) Biochemistry 37, 7708]. The structural model of the catalytic subunit:ATP:PKI((5)(-)(24)) (heat-stable protein kinase inhibitor) ternary complex in the closed conformation suggests that Ser(53) also might be essential for stabilization of the peptide substrate-enzyme complex via a hydrogen bond between the P-site carbonyl in PKI and the Ser(53) side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 12, 849]. To address the importance of the Ser(53) side chain in catalysis, inhibition, and P-site specificity, Ser(53) was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no effect on the steady-state phosphorylation of a peptide substrate (LRRASLG) or on the interaction with physiological inhibitors, including the type-I and -II regulatory subunits and PKI. However, this mutation did affect the P-site specificity; the glycine mutant can more readily phosphorylate a P-site threonine in a peptide substrate (5-6-fold better than wild-type). The proline mutant is compromised catalytically with altered k(cat) and K(m) for both peptide and ATP and with altered sensitivity to both regulatory subunits and PKI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Ser(53) may be required for efficient catalysis, the side chain is not.

Rous sarcoma virus-transformed chicken embryo fibroblasts (RSVCEF), when compared to normal CEF, produce elevated levels of matrix metalloproteinase-2 (MMP-2) that exists in a form free of complexed tissue inhibitor of metalloproteinase-2 (TIMP-2). In order to ascertain whether the increased levels of TIMP-free MMP-2 in RSVCEF cultures are due to diminished expression of TIMP-2 or alterations in TIMP-2 that diminish its MMP-2 binding ability, it was necessary to clone, characterize, and express chicken TIMP-2 cDNA. The TIMP-2 cDNA was cloned from a chick embryo lambda gt11 library by RT-PCR using primers based on amino-acid sequences determined from isolated TIMP-2. The deduced amino acid sequence for chicken TIMP-2 is 81% identical to human TIMP-2; most of the sequence differences lie in the carboxyl terminal portion of chicken TIMP-2. Northern analysis of mRNA levels in CEF and RSVCEF demonstrates that TIMP-2 mRNA levels are increased in RSVCEF. However, TIMP-2 protein levels, relative to proMMP-2 levels, appear to decrease upon transformation and suggest additional control of TIMP-2 at the post-transcriptional level. Addition of recombinantly expressed TIMP-2 to RSVCEF cultures causes a disappearance of TIMP-free (TF) proMMP-2 with a corresponding increase in the TIMP-complexed (TC) proMMP-2 levels, demonstrating that TF proMMP-2 is capable of converting to TC pro-MMP-2 when free TIMP-2 is available. Surprisingly, RSVCEF cultures manifest a TIMP-2 population that is not complexed to MMP-2, despite the coexistence of TIMP-free proMMP-2. Gel-filtration analysis indicates that this uncomplexed TIMP-2 exhibits an apparent molecular weight of 50 kDa, indicating it is not free TIMP-2 and that it exists in transformed cultures in a noncovalent complex with an undefined molecule. Thus transformed cells can alter the TIMP-2/MMP-2 balance by transcriptional and post-translational modifications, yielding a population of inhibitor-free, proteolytically active MMP2.

A variety of invertebrates possess plasma lectins with sialic acid recognition capabilities. One of the best studied of these lectins is limulin, which is a member of the pentraxin family of proteins and is found in the plasma of the American horseshoe crab, Limulus polyphemus. We find that limulin is one of several sialic acid-binding lectins of Limulus plasma and is present at a much lower abundance than Limulus C-reactive protein, the other plasma pentraxin. Limulin was purified by sequential affinity chromatography on phosphorylethanolamine-agarose, which isolates the pentraxins and separates limulin from the other sialic acid-binding lectins of the plasma, followed by fetuin-Sepharose, which binds limulin and separates it from Limulus C-reactive protein, the most abundant pentraxin of the plasma. We show here that limulin is the mediator of the Ca+2-dependent hemolytic activity found in the plasma of Limulus. Plasma that was depleted in the pentraxins by passage over phosphorylethanolamine-agarose or was depleted in the sialic acid-binding lectins by passage over fetuin-Sepharose lacked hemolytic activity. Purified limulin was hemolytic at concentrations of 3-5 nM. The other sialic acid-binding lectins of Limulus plasma and Limulus C-reactive protein were nonhemolytic. Foreign cell cytolysis by limulin represents a novel function for a plasma lectin and is the first documented function for limulin.

Summary: Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin alpha v beta 3. MMP-2 and alpha v beta 3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of alpha v beta 3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind alpha v beta 3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.

Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF) constitute a well-characterized model system for oncogenic transformation, matrix degradation, and cancer invasion. As RSVCEF cultures employ both serine protease and metalloprotease cascades in the process of matrix degradation, they have contributed significantly to understanding the nature and regulation of these molecules involved in invasive cell behavior. RSVCEF produce elevated levels of a matrix metalloprotease-2 (MMP-2) whose hemopexin domain differs from mammalian MMP-2. The majority of MMP-2 produced by RSVCEF is present in a TIMP-free form which enhances its activation, catalytic activity and substrate specificity and therefore its matrix-degrading ability. RSVCEFs also exhibit high levels of urokinase-type plasminogen activator (uPA), which is found in active form in their conditioned medium in complete absence of plasminogen. Recombinantly expressed avian uPA is also in active form, while an active-site mutant of the same maintains its zymogen form, indicating the mechanism of activation of chicken uPA is autocatalytic. A domain and sequence comparison between chicken and human uPA attempts to identify motifs potentially responsible for the zymogen instability of avian uPA and its capability to autoactivate.

The 72-kDa gelatinase/type IV collagenase (MMP-2) is a member of the matrix metalloproteinase (MMP) family of enzymes. This enzyme is known to cleave type IV collagen as well as degrade denatured collagens. However, native interstitial collagens are reportedly resistant to MMP-2 and are thought to be susceptible only to the interstitial collagenases MMP-1 and MMP-8. In this study we report that both human and chicken MMP-2, free of tissue inhibitors of metalloproteinases (TIMPs) are capable of cleaving soluble, triple helical type I collagen generating the 3/4- and 1/4-length collagen fragments characteristic of vertebrate interstitial collagenases. MMP-2 cleaves at the same Gly-Ile/Leu bond in the collagen alpha chains as interstitial collagenases with kcat and Km values similar to that of MMP-1. MMP-2 also is capable of degrading reconstituted type I collagen fibrils. The closely related 92-kDa gelatinase/type IV collagenase (MMP-9) is unable to cleave soluble or fibrillar collagen under identical conditions indicating that the specific collagenolytic activity of MMP-2 is not a general property of gelatinases. That MMP-2, a potent gelatinase, also can cleave fibrillar collagen provides an alternative to the proposal that two enzymes, an interstitial collagenase and a gelatinase, are required for the complete dissolution of stromal collagen during cellular invasion.

Rous sarcoma virus-transformed cultures of chicken embryo fibroblasts (RSVCEF) secrete elevated levels of a 70 kDa progelatinase, an avian form of the 72 kDa matrix metalloproteinase-2 (MMP-2). Affinity-purified preparations of secreted 70 kDa progelatinase are composed of two distinct populations of zymogen: a 70 kDa progelatinase tightly complexed with an avian form of TIMP-2 and a native 70 kDa progelatinase free of any detectable TIMP-2. These two forms of the progelatinase can be separated by Mono Q FPLC in the absence of denaturing agents. The homogeneity of the two separated forms is demonstrated by both SDS-PAGE and nondenaturing, native gel electrophoresis. The purified TIMP-free 70 kDa progelatinase is stable in aqueous conditions and does not spontaneously autoactivate. Treatment of the TIMP-free progelatinase with the organomercurial, p-aminophenylmercuric acetate (APMA), results in rapid (5-60 minutes) autolytic conversion of the 70 kDa progelatinase to 67 kDa, 62 kDa and lower molecular weight forms of the enzyme. APMA treatment of the TIMP-free progelatinase yields a preparation that is enzymatically active with a high specific activity towards a peptide substrate. Identical treatment of TIMP-complexed progelatinase with APMA results in a significantly slower conversion process in which the 70 kDa progelatinase is only 50% converted after 6-24 hours and the specific enzyme activity of the preparation is 8 to 18-fold lower. Purified avian TIMP-2 added to the TIMP-free progelatinase forms a complex with the progelatinase and prevents the rapid autolytic conversion induced by APMA. Comparative analysis of parallel cultures of transformed RSVCEF and normal CEF demonstrates that the transformed cultures contain threefold higher levels of the TIMP-free progelatinase than the normal CEF cultures which produce predominantly TIMP-complexed progelatinase. The presence in transformed cultures of elevated levels of a more readily activated TIMP-free progelatinase, the suppression of its rapid activation by TIMP-2, and the potential effect of the altered balance between TIMP-free and TIMP-complexed 70 kDa progelatinase on the invasive, malignant phenotype, are discussed.

Chicken embryo fibroblasts secrete a 72 kDa progelatinase that displays all of the characteristics of a matrix metalloproteinase. Employing reverse-transcription PCR and degenerate oligonucleotide primers that are specific for two highly conserved sequences found in all matrix metalloproteinases, a DNA fragment specific for the chicken gelatinase was generated. Using this PCR product as a probe, cDNA clones were isolated from a chicken embryo cDNA library and the entire protein coding sequence was determined. The chicken progelatinase is 84% identical, at the amino acid level, with human and mouse 72 kDa progelatinase/type-IV procollagenase, with the greatest degree of similarity occurring in the propeptide and catalytic domains. The avian and mammalian proteinases diverge significantly in the C-terminal, hemopexin-like domain. The last 100 residues of the chicken gelatinase are only 66% identical with mammalian gelatinases. Mouse 72 kDa progelatinase, however, does not diverge significantly (> 98% identity) from human progelatinase in the hemopexin-like domain. The divergence in this domain of the chicken progelatinase may explain some of the distinct catalytic and inhibitory properties of the 72 kDa chicken progelatinase. Northern-blot analysis reveals that steady-state levels of the chicken progelatinase mRNA are increased 5-fold upon malignant transformation of chicken embryo fibroblasts with Rous sarcoma virus (RSV) and 3-fold by treatment with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA). This represents the first reported cloning of an avian matrix metalloproteinase. The increased expression of the chicken progelatinase by RSV transformation and the tumour promoter PMA suggests that the progelatinase is regulated differently in chicken cells.

Chicken embryo fibroblasts (CEF) produce a 70kDa progelatinase, a member of the matrix metalloproteinase family, and secrete elevated levels of the enzyme upon transformation by Rous sarcoma virus (RSV). This enzyme can be purified by affinity chromatography complexed with a 21kDa tissue inhibitor of metalloproteinases (TIMP)-like molecule. Gel-filtration of the purified progelatinase suggests the presence of a mixed population of enzyme: a TIMP-complexed and a TIMP-free progelatinase. These two species were separated by Mono Q FPLC in the absence of denaturants. Quantitation of the purified progelatinase reveals that the transformed RSVCEF produce more TIMP-free enzyme than the normal CEF. Native PAGE analysis indicates that purified TIMP-free progelatinase is capable of binding to TIMP and generating a TIMP-complexed progelatinase. Treatment of the TIMP-free gelatinase with organomercurials results in a rapid conversion to the active 62kDa species and indicates that the TIMP-free progelatinase is more susceptible to activation than the TIMP-complexed progelatinase.

Chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSVCEF) secrete a 70-kDa metallo-gelatinase at elevated levels over that of normal CEF. The 70-kDa enzyme has been purified from RSVCEF conditioned medium and represents 1-3% of the total protein in the RSVCEF conditioned medium. A 22-kDa protein, which appears to be the avian form of the tissue inhibitor of metalloproteases (TIMP), is co-isolated in association with the 70-kDa enzyme and can be separated from the enzyme by gel filtration carried out under denaturing conditions. The isolated 70-kDa species is in the zymogen form. It can be activated by treatment with the organomercurial, p-aminophenylmercuric acetate (APMA), yielding a 62-kDa active species derived by an apparent autoproteolytic cleavage from the 70-kDa proenzyme as determined by both substrate gel analysis and immunoblots using a monospecific antibody to the 70-kDa proenzyme. The proenzyme is poorly activated by trypsin and not activated by plasmin. The APMA-activated enzyme rapidly degrades denatured collagens but under identical conditions is unable to degrade native collagens, including basement membrane type IV collagen. Only at very high enzyme to substrate ratios (1:2) will native type IV collagen be hydrolyzed. Partial N-terminal amino acid sequencing of both the 70-kDa proenzyme and the 62-kDa active enzyme indicates that the avian enzyme is a member of the matrix metalloprotease family (MMP-2). When CEF cultures, infected with a temperature sensitive mutant of RSV, conditional for the expression of the transforming src oncogene, were incubated at the permissive and nonpermissive temperatures, differential levels of the 70-kDa enzyme were produced in direct proportion to the functioning of the src oncogene.

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.