In vivo cleaved CDCP1 promotes early tumor dissemination via complexing with activated ß1 integrin and induction of FAK/PI3K/Akt motility signaling.

Specific cleavage of the transmembrane molecule, CUB domain-containing protein-1 (CDCP1), by plasmin-like serine proteases induces outside-in signal transduction that facilitates early stages of spontaneous metastasis leading to tumor cell intravasation, namely cell escape from the primary tumor, stromal invasion and transendothelial migration. We identified active ß1 integrin as a biochemical and functional partner of the membrane-retained 70-kDa CDCP1 fragment, newly generated from its full-length 135-kDa precursor though proteolytic cleavage by serine proteases. Both in cell cultures and in live animals, active ß1 integrin complexed preferentially with functionally activated, phosphorylated 70-kDa CDCP1. Complexing of ß1 integrin the 70-kDa with CDCP1 fragment induced intracellular phosphorylation signaling, involving focal adhesion kinase-1 (FAK) and PI3 kinase (PI3K)-dependent Akt activation. Thus, inhibition of FAK/PI3K activities by specific inhibitors as well as short-hairpin RNA downregulation of ß1 integrin significantly reduced FAK/Akt phosphorylation under conditions where CDCP1 was processed by serine proteases, indicating that FAK/PI3K/Akt pathway operates downstream of cleaved CDCP1 complexed with ß1 integrin. Furthermore, this complex-dependent signaling correlated positively with high levels of tumor cell intravasation and dissemination. Correspondingly, abrogation in vivo of CDCP1 cleavage either by unique cleavage-blocking monoclonal antibody 10-D7 or by inhibition of proteolytic activity of plasmin-like serine proteases with aprotinin prevented ß1 integrin/CDCP1 complexing and downstream FAK/Akt signaling concomitant with significant reduction of stromal invasion and spontaneous metastasis. Therefore, ß1 integrin appears to serve as a motility-regulating partner mediating cross-talk between proteolytically cleaved, membrane-retained CDCP1 and members of FAK/PI3K/Akt pathway. This CDCP1 cleavage-induced signaling cascade constitutes a unique mechanism, independent of extracellular matrix remodeling, whereby a proteolytically cleaved CDCP1 regulates in vivo locomotion and metastasis of tumor cells through ß1 integrin partnering. Our findings indicate that CDCP1 cleavage, occurring at the apex of a ß1 integrin/FAK/PI3K/Akt signaling cascade, may represent a therapeutic target for CDCP1-positive cancers.

Blocking of CDCP1 cleavage in vivo prevents Akt-dependent survival and inhibits metastatic colonization through PARP1-mediated apoptosis of cancer cells.

The CUB domain-containing protein-1 (CDCP1) is a transmembrane molecule that has recently been implicated in cancer progression. In this study we have established a novel mechanism for initiation of CDCP1-mediated signaling in vivo and demonstrated that specific 135→70-kDa processing of cell-surface CDCP1 by extracellular serine proteases is a prerequisite for CDCP1-dependent survival of cancer cells during metastasis. The in vivo cleavage of CDCP1 triggers a survival program involving recruitment of Src and PKCδ, Src-mediated phosphorylation of cell-surface-retained 70-kDa CDCP1, activation of Akt and suppression of PARP1-induced apoptosis. We demonstrate in vivo that phosphorylated Src, PKCδ and Akt all constitute activated elements of a CDCP1-signaling axis during tissue colonization of tumor cells. Preventing in vivo cleavage of CDCP1 with unique anti-CDCP1 antibodies, serine protease inhibitors or genetic modulation of the cleavage site in the CDCP1 molecule completely abrogated survival signaling associated with the 70-kDa CDCP1, and induced PARP1 cleavage and PARP1-mediated apoptosis, ultimately resulting in substantial inhibition of tissue colonization by tumor cells. The lack of CDCP1 cleavage in the lung tissue of plasminogen-knockout mice along with a coordinated reduction in tumor cell survival in a lung retention model, and importantly rescue of both by in vivo supplied plasmin, indicated that plasmin is the crucial serine protease executing in vivo cleavage of cell-surface CDCP1 during early stages of lung colonization. Together, our findings indicate that in vivo blocking of CDCP1 cleavage upstream from CDCP1-induced pro-survival signaling provides a potential mechanism for therapeutic intervention into metastatic disease.

Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MMP should exist as oligomers on the cell surface. To better understand the functions of MT1-MMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP. Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface. The presence of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells into the mature proteinase capable of activating pro-MMP-2 and stimulating cell locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydroxamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independent, alternative pathway of MT1-MMP activation in breast carcinoma cells. The expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part of MT1-MMP and the intracellular milieu.

Growth factor-induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen.

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.

The isolation, characterization, and molecular cloning of a 75-kDa gelatinase B-like enzyme, a member of the matrix metalloproteinase (MMP) family. An avian enzyme that is MMP-9-like in its cell expression pattern but diverges from mammalian gelatinase B in sequence and biochemical properties.

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.

Binding of alpha2-macroglobulin and limulin: regulation of the plasma haemolytic system of the American horseshoe crab, Limulus.

The mediator of haemolysis in the plasma of the horseshoe crab, Limulus polyphemus, is limulin, a sialic acid-binding lectin. The haemolytic activity of limulin is inhibited by thiol ester-reacted forms of Limulus alpha(2)-macroglobulin, the third-most abundant protein of the plasma. Limulus alpha(2)-macroglobulin that has experienced cleavage of its internal thiol ester bond, consequent to reaction with proteases, or with the small primary amine, methylamine, reduces the haemolytic activity of limulin when present at molar excesses that approximate the relative concentrations of these two proteins in the plasma. Native, unreacted Limulus alpha(2)-macroglobulin has no effect on the haemolytic activity of limulin. Limulin binds thiol ester-reacted forms of Limulus alpha(2)-macroglobulin both in a solid-phase assay and in solution with an avidity 10-25 times higher than native, unreacted Limulus alpha(2)-macroglobulin. Protease-reacted alpha(2)-macroglobulin functions as a marker for the presence of foreign proteases in the blood of Limulus, and thus of pathogenic organisms that release proteases as facilitators of invasion and pathogenicity. Modulation of the haemolytic system represents a novel function for alpha(2)-macroglobulin.

Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis.

A monoclonal antibody (mAb), 50-6, generated by subtractive immunization, was found to specifically inhibit in vivo metastasis of a human epidermoid carcinoma cell line, HEp-3. The cDNA of the cognate antigen of mAb 50-6 was isolated by a modified eukaryotic expression cloning protocol from a HEp-3 library. Sequence analysis identified the antigen as PETA-3/CD151, a recently described member of the tetraspanin family of proteins. The cloned antigen was also recognized by a previously described antimetastatic antibody, mAb 1A5. Inhibition of HEp-3 metastasis by the mAbs could not be attributed to any effect of the antibodies on tumor cell growth in vitro or in vivo. Rather, the antibodies appeared to inhibit an early step in the formation of metastatic foci. In a chemotaxis assay, HEp-3 migration was blocked by both antibodies. HeLa cells transfected with and overexpressing PETA-3/CD151 were more migratory than control transfectants expressing little CD151. The increase in HeLa migration was inhibitable by both mAb 50-6 and mAb 1A5. PETA-3 appears not to be involved in cell attachment because adhesion did not correlate with levels of PETA-3 expression and was unaffected by mAb 50-6 or mAb 1A5. The ability of PETA-3 to mediate cell migration suggests a mechanism by which this protein may influence metastasis. These data identify PETA-3/CD151 as the first member of the tetraspanin family to be linked as a positive effector of metastasis.

Activation of proMMP-9 by a plasmin/MMP-3 cascade in a tumor cell model. Regulation by tissue inhibitors of metalloproteinases.

To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells that were induced to produce MMP-9 over a 200-fold concentration range (0.03 to 8.1 nM). The secreted levels of TIMPs in all the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether proMMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but only via an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous plasminogen activator (uPA), is not an efficient activator of proMMP-9. Plasmin, however, is very efficient at generating active MMP-3 from exogenously added proMMP-3. The activated MMP-3, when its concentration exceeds that to TIMP, becomes a potent activator of proMMP-9. Addition to the cultures of already-activated MMP-3 relinquishes the requirement for plasminogen and proMMP-3 additions and results in direct activation of the endogenous proMMP-9. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodeling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9-blocking monoclonal antibody.

Alpha2-macroglobulin: an evolutionarily conserved arm of the innate immune system.

All animals and plants have immune systems that protect them from the diversity of pathogens that would otherwise threaten their survival. The different components of the immune system may inactivate the pathogens themselves or promote the inactivation and clearance of toxic products produced by the pathogens. An important category of virulence factors of bacterial and prokaryotic pathogens are the proteases, which act to facilitate the invasion of the pathogens and to promote their destructive growth in the host organism. The present review concentrates on the comparative biology of an evolutionarily conserved arm of the immune system, the protein, alpha2-macroglobulin. alpha2-Macroglobulin is an abundant protein of the plasma of vertebrates and members of several invertebrate phyla and functions as a broad-spectrum protease-binding protein. Protease-conjugated alpha2-macroglobulin is selectively bound by cells contacting the body fluids and alpha2-macroglobulin and its protease cargo are then internalized and degraded in secondary lysosomes of those cells. In addition to this function as an agent for protease clearance, alpha2-macroglobulin binds a variety of other ligands, including several peptide growth factors and modulates the activity of a lectin-dependent cytolytic pathway in arthropods.

Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion.

Matrix metalloproteinase-9 (MMP-9) may play a critical catalytic role in tissue remodeling in vivo, but it is secreted by cells as a stable, inactive zymogen, pro-MMP-9, and requires activation for catalytic function. A number of proteolytic enzymes activate pro-MMP-9 in vitro, but the natural activator(s) of MMP-9 is unknown. To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells (MDA-MB-231 breast carcinoma cells) that were induced to produce MMP-9 over a 200-fold concentration range (0.03-8.1 nM). The levels of tissue inhibitors of metalloproteinase (TIMPs) in the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the MDA-MB-231 cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether pro-MMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but through an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous urokinase-type plasminogen activator, is not an efficient activator of pro-MMP-9, neither the secreted pro-MMP-9 nor the very low levels of pro-MMP-9 associated with intact cells. Although plasmin can proteolytically process pro-MMP-9, this limited action does not yield an enzymatically active MMP-9, nor does it cause the MMP-9 to be more susceptible to activation. Plasmin, however, is very efficient at generating active MMP-3 (stromelysin-1) from exogenously added pro-MMP-3. The activated MMP-3 becomes a potent activator of the 92-kDa pro-MMP-9, yielding an 82-kDa species that is enzymatically active in solution and represents up to 50-75% conversion of the zymogen. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to both degrade extracellular matrix and transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodelling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9 blocking monoclonal antibody.

Co-inoculation of human and murine carcinoma cells induces reciprocal suppression of metastasis by both cell lines.

The interactions of two cell lines having different metastatic properties, and the subsequent effects on dissemination were investigated using the chicken embryo metastasis assay. The highly aggressive human epidermoid cell line HEp-3 was tested alone or mixed with the mouse colon carcinoma cell line CL26 in this assay. When inoculated individually, each cell line forms experimental metastases in the chicken embryo, but only the HEp-3 cells give rise to spontaneous metastases. In embryos co-inoculated with both cell lines there was an overall reduction in metastatic burden in both the spontaneous and experimental metastasis assays. Furthers studies revealed that CL26 cells, when co-inoculated with HEp-3 cells did not acquire the ability to spontaneously metastasize. However, in the presence of CL26 cells, spontaneous HEp-3 metastasis was reduced. Intravenous co-inoculation of HEp-3 and CL26 cells also resulted in a reciprocal suppression of experimental metastasis by both cell lines. These studies demonstrate that the interactions of adjacent, phenotypically different tumor cells can have a suppressive effect on dissemination of one or both cell types.

Alpha2-macroglobulin does not function as a C3 homologue in the plasma hemolytic system of the American horseshoe crab, Limulus.

A major problem of comparative immunology is the characterization of the internal defense systems that lyse foreign cells, such as bacteria and other microbial pathogens that have gained entry into the body. The plasma cytolytic system of the American horseshoe crab, Limulus polyphemus, is sensitive to treatment with methylamine, which inactivates the abundant plasma defense protein alpha2-macroglobulin. This has been interpreted to mean that alpha2-macroglobulin plays an important role in hemolysis, analogous to the role of complement component C3 of the mammalian complement system (Enghild et al., 1990). Sensitivity to methylamine has been suggested to reflect an evolutionary homology with the plasma cytolytic system of mammals, in which the complement system is inactivated by the reaction of methylamine with complement components C3 and C4. C3, C4 and alpha2-macroglobulin contain an internal thiol ester bond linking cysteinyl and glutamic acid residues and methylamine inactivates all three proteins by reaction with the thiol-esterified glutamic acid. However, we have recently shown that the principal effector of hemolysis in Limulus is the plasma lectin, limulin (Armstrong et al., 1996). In this article we show that native, unreacted alpha2-macroglobulin is not involved directly in hemolysis but instead that methylamine-reacted alpha2-macroglobulin inhibits the hemolytic activity of limulin. Thus the thiol ester proteins alpha2-macroglobulin and C3 operate very differently in the hemolytic systems of Limulus and mammals and are not functionally homologous. Limulus alpha2-macroglobulin functions indirectly in hemolysis: its inactivation yields an inhibitory molecule for limulin-mediated hemolysis.

Cloning, expression, and characterization of chicken tissue inhibitor of metalloproteinase-2 (TIMP-2) in normal and transformed chicken embryo fibroblasts.

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.

Comparison of Limulus alpha-macroglobulin with human alpha2-macroglobulin: thiol ester characterization, subunit organization, and conformational change.

The properties of the thiol ester-containing alpha-macroglobulin (alphaM) from the horseshoe crab (Limulus polyphemus) have been compared with those of the human analogue (alpha2M). Thiol ester accessibility was more restricted in Limulus alphaM than in human alpha2M. Fluorescent probes attached to the thiol ester cysteine indicated very similar local environments in the cleaved state of the two alphaMs. The separation between the two thiol ester cysteines in the cleaved state, determined by fluorescence resonance energy transfer, was also very similar for the two alphaMs. Differences were found in the oligomerization state and conformational changes of the two proteins. Whereas human alpha2M appears to be exclusively a dimer of dimers, Limulus alphaM can exist in both tetrameric and dimeric forms, although with marked preference for the dimer. Conformational change within a dimeric trapping unit, monitored by 6-(p-toluidino)-2-napthalene-sulfonic acid fluorescence change, showed that each monomer of the Limulus alphaM dimer appears to change conformation independently, whereas human alpha2M requires both thiol esters within a functional unit to be cleaved before the conformational change occurs. Taken together, these findings indicate that, whereas individual thiol esters in both types of alphaM are similar in properties, differences in subunit-subunit interaction result both in differences in state of oligomerization and in cooperativity of conformational change, which may reflect a fundamentally different organization of the subunits within a dimer in the two alphaMs.

Molecular cloning of Limulus alpha 2-macroglobulin.

The American horseshoe crab Limulus polyphemus contains alpha 2-macroglobulin (alpha 2M) in the hemolymph plasma and hemocytes. alpha 2M from Limulus shows many of the typical characteristics of mammalian alpha 2M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian alpha 2M receptor. Additionally, Limulus alpha 2M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulus alpha 2M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian alpha 2Ms (28-29% identity) and contains common features found in mammalian alpha 2Ms. a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24-105) has no sequence similarity with those of mammalian alpha 2Ms, and it is structurally related to that of the human complement factor C8 chain, consistent with a role for Limulus alpha 2M in host defense. The component sugar analysis of Limulus alpha 2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian alpha 2M. However, unlike mammalian alpha 2M, no sialic acid was detected in Limulus alpha 2M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian alpha 2M. Expression of alpha 2M was detected in hemocytes, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against alpha 2M indicated the presence of the alpha 2M in large granules. Trypsin-treated Limulus alpha 2M, but not the native alpha 2M, displaced methylamine-treated human 125I-alpha 2M from the human alpha 2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.

A cytolytic function for a sialic acid-binding lectin that is a member of the pentraxin family of proteins.

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.

Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3.

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.

What structure and function of avian plasminogen activator and matrix metalloproteinase-2 reveal about their counterpart mammalian enzymes, their regulation and their role in tumor 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.