Mammalian BMP-1/Tolloid-related metalloproteinases, including novel family member mammalian Tolloid-like 2, have differential enzymatic activities and distributions of expression relevant to patterning and skeletogenesis.

Vertebrate bone morphogenetic protein 1 (BMP-1) and Drosophila Tolloid (TLD) are prototypes of a family of metalloproteases with important roles in various developmental events. BMP-1 affects morphogenesis, at least partly, via biosynthetic processing of fibrillar collagens, while TLD affects dorsal-ventral patterning by releasing TGFbeta-like ligands from latent complexes with the secreted protein Short Gastrulation (SOG). Here, in a screen for additional mammalian members of this family of developmental proteases, we identify novel family member mammalian Tolloid-like 2 (mTLL-2) and compare enzymatic activities and expression domains of all four known mammalian BMP-1/TLD-like proteases [BMP-1, mammalian Tolloid (mTLD), mammalian Tolloid-like 1 (mTLL-1), and mTLL-2]. Despite high sequence similarities, distinct differences are shown in ability to process fibrillar collagen precursors and to cleave Chordin, the vertebrate orthologue of SOG. As previously demonstrated for BMP-1 and mTLD, mTLL-1 is shown to specifically process procollagen C-propeptides at the physiologically relevant site, while mTLL-2 is shown to lack this activity. BMP-1 and mTLL-1 are shown to cleave Chordin, at sites similar to procollagen C-propeptide cleavage sites, and to counteract dorsalizing effects of Chordin upon overexpression in Xenopus embryos. Proteases mTLD and mTLL-2 do not cleave Chordin. Differences in enzymatic activities and expression domains of the four proteases suggest BMP-1 as the major Chordin antagonist in early mammalian embryogenesis and in pre- and postnatal skeletogenesis.     

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.     

Normal human keratinocytes bind to the alpha3LG4/5 domain of unprocessed laminin-5 through the receptor syndecan-1

Basal keratinocytes of the epidermis adhere to their underlying basement membrane through a specific interaction with laminin-5, which is composed by the association of alpha3, beta3, and gamma2 chains. Laminin-5 has the ability to induce either stable cell adhesion or migration depending on specific processing of different parts of the molecule. One event results in the cleavage of the carboxyl-terminal globular domains 4 and 5 (LG4/5) of the alpha3 chain. In this study, we recombinantly expressed the human alpha3LG4/5 fragment in mammalian cells, and we show that this fragment induces adhesion of normal human keratinocytes and fibrosarcoma-derived HT1080 cells in a heparan- and chondroitin sulfate-dependent manner. Immunoprecipitation experiments with Na2 35SO4-labeled keratinocyte and HT1080 cell lysates as well as immunoblotting experiments revealed that the major proteoglycan receptor for the alpha3LG4/5 fragment is syndecan-1. Syndecan-4 from keratinocytes also bound to alpha3LG4/5. Furthermore we could show for the first time that unprocessed laminin-5 specifically binds syndecan-1, while processed laminin-5 does not. These results demonstrate that the LG4/5 modules within unprocessed laminin-5 permit its cell binding activity through heparan and chondroitin sulfate chains of syndecan-1 and reinforce previous data suggesting specific properties for the precursor molecule.     

Laminin-5 suppresses chondrogenic differentiation of murine teratocarcinoma cell line ATDC5

Laminin-5 is an important basement membrane protein that regulates cell adhesion and motility. It was previously found that the gamma2 chain of laminin-5 is transiently expressed in embryonic cartilage. This suggests a possible role of laminin-5 in chondrogenesis. Here, we examined this possibility using the murine teratocarcinoma cell line ATDC5. ATDC5 cells transiently and weakly expressed laminin-5 when they were stimulated for differentiation. Exogenous laminin-5 in either insoluble or soluble form strongly inhibited the differentiation phenotypes, i.e. formation of cartilaginous cell aggregates and production of chondrogenic marker proteins through its integrin-binding domain LG3 in the alpha3 chain. Laminin-5 had no effect on cell growth. In addition, we found that the laminin-5 with the 105-kDa, processed gamma2 chain suppressed differentiation more strongly than one with the 150-kDa gamma2 chain. This indicated that the proteolytic processing of gamma2 chain regulated the activity of laminin-5. However, a gamma2 chain short arm fragment had no effect on the chondrogenesis, and it rather suppressed the differentiation at excessive concentrations. These results suggest that laminin-5 and its processing modulate chondrogenic differentiation during development.     

Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of bone morphogenetic protein 1/tolloid-like metalloproteinases

Bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD), two proteinases encoded by Bmp1, provide procollagen C-proteinase (pCP) activity that converts procollagens I to III into the major fibrous components of mammalian extracellular matrix (ECM). Yet, although Bmp1(-/-) mice have aberrant collagen fibrils, they have residual pCP activity, indicative of genetic redundancy. Mammals possess two additional proteinases structurally similar to BMP-1 and mTLD: the genetically distinct mammalian Tolloid-like 1 (mTLL-1) and mTLL-2. Mice lacking the mTLL-1 gene Tll1 are embryonic lethal but have pCP activity levels similar to those of the wild type, suggesting that mTLL-1 might not be an in vivo pCP. In vitro studies have shown BMP-1 and mTLL-1 capable of cleaving Chordin, an extracellular antagonist of BMP signaling, suggesting that these proteases might also serve to modulate BMP signaling and to coordinate the latter with ECM formation. However, in vivo evidence of roles for BMP-1 and mTLL-1 in BMP signaling in mammals is lacking. To remove functional redundancy obscuring the in vivo functions of BMP-1-related proteases in mammals, we here characterize Bmp1 Tll1 doubly null mouse embryos. Although these appear morphologically indistinguishable from Tll1(-/-) embryos, biochemical analysis of cells derived from doubly null embryos shows functional redundancy removed to an extent enabling us to demonstrate that (i) products of Bmp1 and Tll1 are responsible for in vivo cleavage of Chordin in mammals and (ii) mTLL-1 is an in vivo pCP that provides residual activity observed in Bmp1(-/-) embryos. Removal of functional redundancy also enabled use of Bmp1(-/-) Tll1(-/-) cells in a proteomics approach for identifying novel substrates of Bmp1 and Tll1 products.     

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1)

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.     

Recombinant human laminin-5 domains. Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha3beta1 integrin binding

Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The alpha3' chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type alpha3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, beta3'and gamma2', produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the alpha3' chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1-3, but not the single chains, supported binding of soluble alpha3beta1 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with alpha3beta1 integrin. The N-glycosylation sites in wild-type alpha3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor alpha3beta1 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4-5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the alpha3'LG1-3 chain showed an increased binding affinity for alpha3beta1 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with alpha3beta1 integrin.     

Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin-3 from placenta

Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.     

The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart.

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1-/- embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.     

Isolation and activity of proteolytic fragment of laminin-5 alpha3 chain

Laminin-5 (alpha3beta3gamma2) is an important component of epithelial basement membranes. The 190-kDa alpha3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa alpha3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the alpha3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln(1337)-Asp(1338) in the spacer region between the G3 and G4 domains. The G4-G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration.     

Bone morphogenetic protein 1 is an extracellular processing enzyme of the laminin 5 gamma 2 chain

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (alpha3beta3gamma2) within the alpha3 and gamma2 chains (). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first alpha3 chain cleavage (200-l65 kDa alpha3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa alpha3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The gamma chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the gamma2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (). Recombinant BMP-1 cleaves gamma2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant gamma2 short arm. alpha3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin alpha3 and gamma2 chains to be substrates for BMP-1 in vitro. We speculate that gamma2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.     

Bone morphogenetic protein-1/Tolloid-like proteinases process dentin matrix protein-1

Bone morphogenetic protein-1 (BMP-1)/Tolloid-like metalloproteinases play key roles in formation of mammalian extracellular matrix (ECM), through the biosynthetic conversion of precursor proteins into their mature functional forms. These proteinases probably play a further role in formation of bone through activation of transforming growth factor beta-like BMPs. Dentin matrix protein-1 (DMP1), deposited into the ECM during assembly and involved in initiating mineralization of bones and teeth, is thought to undergo proteolysis in vivo to generate functional cleavage fragments found in extracts of mineralized tissues. Here, we have generated recombinant DMP1 and demonstrate that it is cleaved, to varying extents, by all four mammalian BMP-1/Tolloid-like proteinases, to generate fragments similar in size to those previously isolated from bone. Consistent with possible roles for the BMP-1/Tolloid-like proteinases in the physiological processing of DMP1, NH2-terminal sequences of products generated by BMP-1 cleavage of DMP1 match those predicted from processing at the predicted DMP1 site that shows greatest cross-species conservation of sequences. Moreover, fibroblasts derived from mouse embryos homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-like proteinases appear to be deficient in processing of DMP1. Thus, a further role for BMP-1-Tolloid-like proteinases in formation of mineralized tissues is indicated, via proteolytic processing of DMP1.     

Localization of laminin α3B chain in vascular and epithelial basement membranes of normal human tissues and its down-regulation in skin cancers

The basement membrane (BM) proteins laminins, which consist of alpha, beta and gamma chains, play critical roles in the maintenance of tissue structures. One of laminin alpha chains, alpha3 has two isoforms, the truncated form alpha3A and the full-sized form alpha3B. In contrast to alpha3A laminins, little is known about alpha3B laminins. To show the histological distribution of the laminin alpha3B chain, we prepared alpha3B-specific monoclonal antibodies. Immunohistochemical analysis showed that the alpha3B chain was colocalized with the alpha3A, beta3 and gamma2 chains in the epithelial BMs of the skin, esophagus, breast and lung, suggesting the presence of laminin-3B32 (laminin-5B) and laminin-3A32 (laminin-5A). In the lung alveoli, laminin-3B32 was dominant over laminin-3A32, but vice versa in other epithelial BMs. In contrast, the BMs of blood vessels including capillaries were strongly positive for alpha3B, but almost or completely negative for alpha3A, beta3 and gamma2. alpha3B was colocalized with beta1 and gamma1 in these BMs. The alpha3B chain was scarcely detected in the vessels of malignant skin cancers, though the gamma2 and beta3 chains were highly expressed in the cancer cells. These results strongly suggest that the laminin alpha3B chain is widely expressed in vascular BMs of normal tissues, probably as laminin-3B11/3B21 (laminin-6B/7B).     

Bone morphogenetic protein-1 processes probiglycan

Bone morphogenetic protein-1 (BMP-1) is a metalloprotease that plays important roles in regulating the deposition of fibrous extracellular matrix in vertebrates, including provision of the procollagen C-proteinase activity that processes the major fibrillar collagens I-III. Biglycan, a small leucine-rich proteoglycan, is a nonfibrillar extracellular matrix component with functions that include the positive regulation of bone formation. Biglycan is synthesized as a precursor with an NH(2)-terminal propeptide that is cleaved to yield the mature form found in vertebrate tissues. Here, we show that BMP-1 cleaves probiglycan at a single site, removing the propeptide and producing a biglycan molecule with an NH(2) terminus identical to that of the mature form found in tissues. BMP-1-related proteases mammalian Tolloid and mammalian Tolloid-like 1 (mTLL-1) are shown to have low but detectable levels of probiglycan-cleaving activity. Comparison shows that wild type mouse embryo fibroblasts (MEFs) produce only fully processed biglycan, whereas MEFs derived from embryos homozygous null for the Bmp1 gene, which encodes both BMP-1 and mammalian Tolloid, produce predominantly unprocessed probiglycan, and MEFs homozygous null for both the Bmp1 gene and the mTLL-1 gene Tll1 produce only unprocessed probiglycan. Thus, all detectable probiglycan-processing activity in MEFs is accounted for by the products of these two genes.     

Regulation of biological activity and matrix assembly of laminin-5 by COOH-terminal, LG4-5 domain of alpha3 chain

The basement membrane protein laminin-5 (LN5; alpha3beta3gamma2) undergoes specific proteolytic processing of the 190-kDa alpha3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4-5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa alpha3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type alpha3 chain was completely cleaved, whereas the mutated alpha3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4-5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa alpha3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4-5 from the 190-kDa alpha3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4-5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4-5 synergistically enhances integrin signaling as it is released from the precursor LN5.     

Bone morphogenetic protein-1/tolloid-related metalloproteinases process osteoglycin and enhance its ability to regulate collagen fibrillogenesis

The mammalian bone morphogenetic protein-1 (BMP-1)/Tolloid-related metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating the mineralization of hard tissue ECMs. They also have been shown to activate several members of the transforming growth factor-beta superfamily, and may serve to coordinate such activation with formation of the ECM in morphogenetic events. Osteoglycin (OGN), a small leucine-rich proteoglycan with unclear functions, is found in cornea, bone, and other tissues, and appears to undergo proteolytic processing in vivo. Here we have successfully generated recombinant OGN and have employed it to demonstrate that a pro-form of OGN is processed to varying extents by all four mammalian BMP-1/Tolloid-like proteinases, to generate a 27-kDa species that corresponds to the major form of OGN found in cornea. Moreover, whereas wild-type mouse embryo fibroblasts (MEFs) produce primarily the processed, mature form of OGN, MEFs homozygous null for genes encoding three of the four mammalian BMP-1/Tolloid-related proteinases produce only unprocessed pro-OGN. Thus, all detectable pro-OGN processing activity in MEFs is accounted for by products of these genes. We also demonstrate that both pro- and mature OGN can regulate type I collagen fibrillogenesis, and that processing of the prodomain by BMP-1 potentiates the ability of OGN to modulate the formation of collagen fibrils.     

Biosynthetic processing of the Pro-alpha1(V)Pro-alpha2(V)Pro-alpha3(V) procollagen heterotrimer

Type V collagen is a quantitatively minor fibrillar collagen comprised of different chain compositions in different tissues. The most widely distributed form, an alpha1(V)2alpha2(V) heterotrimer, regulates the physical properties of type I/V heterotypic collagen fibrils via partially processed NH2-terminal globular sequences. A less characterized alpha1(V)alpha2(V)alpha3(V) heterotrimer has a much more limited distribution of expression and unknown function(s). We characterized the biosynthetic processing of pro-alpha1(V)2pro-alpha2(V) procollagen previously and showed it to differ in important ways from biosynthetic processing of the major fibrillar procollagens I-III. Here we have successfully produced recombinant pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers. We use these, and mouse embryo fibroblasts doubly homozygous null for the Bmp1 gene, which encodes the metalloproteinase bone morphogenetic protein-1 (BMP-1), and for a gene encoding the closely related metalloproteinase mammalian Tolloid-like 1, to characterize biosynthetic processing of pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers, thus completing characterization of type V collagen biosynthetic processing. Whereas pro-alpha1(V) and pro-alpha2(V) processing in pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers is similar to that which occurs in pro-alpha1(V)2pro-alpha2(V) heterotrimers, the processing of pro-alpha3(V) by BMP-1 occurs at an unexpected site within NH2-terminal globular sequences. We also demonstrate that, despite similarities in NH2-terminal domain structures, pro-alpha2(V) NH2-terminal globular sequences are not cleaved by ADAMTS-2, the metalloproteinase that cleaves the N-propeptides of the major fibrillar procollagen chains.     

Short arm region of laminin-5 gamma2 chain: structure, mechanism of processing and binding to heparin and proteins.

Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4-6, that flank a globular L4m module containing the cleavage site. Recombinant analysis of L4m, which includes an additional imperfect LE module essential for proper folding, demonstrated an unusual pattern of disulfide bonding. These connectivities prevented the release of gamma2LE1-3L4 m after BMP-1 cleavage which required in addition disulfide reshuffling by isomerases. The liberated segment bound through its L4 m module to heparin, nidogen-1, fibulin-1 and fibulin-2. A further heparin/sulfatide-binding site could be attributed to some arginine residues in module LE1. The gamma2LE4-6 segment remaining in processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo.     

Structural requirement of carboxyl-terminal globular domains of laminin alpha 3 chain for promotion of rapid cell adhesion and migration by laminin-5

The basement membrane protein laminin-5, a heterotrimer of laminin alpha3, beta3, and gamma2 chains, potently promotes cellular adhesion and motility. It has been supposed that the carboxyl-terminal globular region of the alpha3 chain consisting of five distinct domains (G1 to G5) is important for its interaction with integrins. To clarify the function of each G domain, we transfected cDNAs for the full-length (wild type (WT)) and five deletion derivatives (DeltaGs) of the alpha3 chain into human fibrosarcoma cell line HT1080, which expressed and secreted the laminin beta3 and gamma2 chains but not the alpha3 chain. The transfectants with the alpha3 chain cDNAs lacking G5 (DeltaG(5)), G4-5 (DeltaG(4-5)), G3-5 (DeltaG(3-5)), and G2-5 (DeltaG(2-5)) secreted laminin-5 variants at levels comparable to that with WT cDNA. However, the transfectant with the cDNA without any G domains (DeltaG(1-5)) secreted little laminin-5, suggesting that the G domains are essential for the efficient assembly and secretion of the heterotrimer alpha3beta3gamma2. The transfectants with WT, DeltaG(5), and DeltaG(4-5) cDNAs survived in serum-free medium longer than those with DeltaG(3-5), DeltaG(2-5), and DeltaG(1-5) cDNAs. The transfectants with WT, DeltaG(5), and DeltaG(4-5) cDNAs secreted apparently the same size of laminin-5, which lacked G4 and G5 due to proteolytic cleavage between G3 and G4, and these laminin-5 forms potently promoted integrin alpha(3)beta(1)-dependent cell adhesion and migration. However, the laminin-5 forms of DeltaG(3-5) and DeltaG(2-5) hardly promoted the cell adhesion and motility. These findings demonstrate that the G3 domain, but not the G4 and G5 domains, of the alpha3 chain is essential for the potent promotion of cell adhesion and motility by laminin-5.