Collagen VI Microfibril Formation Is Abolished by an α2(VI) von Willebrand Factor Type A Domain Mutation in a Patient with Ullrich Congenital Muscular Dystrophy

Collagen VI is an extracellular protein that most often contains the three genetically distinct polypeptide chains, α1(VI), α2(VI), and α3(VI), although three recently identified chains, α4(VI), α5(VI), and α6(VI), may replace α3(VI) in some situations. Each chain has a triple helix flanked by N- and C-terminal globular domains that share homology with the von Willebrand factor type A (VWA) domains. During biosynthesis, the three chains come together to form triple helical monomers, which then assemble into dimers and tetramers. Tetramers are secreted from the cell and align end-to-end to form microfibrils. The precise molecular mechanisms responsible for assembly are unclear. Mutations in the three collagen VI genes can disrupt collagen VI biosynthesis and matrix organization and are the cause of the inherited disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. We have identified a Ullrich congenital muscular dystrophy patient with compound heterozygous mutations in α2(VI). The first mutation causes skipping of exon 24, and the mRNA is degraded by nonsense-mediated decay. The second mutation is a two-amino acid deletion in the C1 VWA domain. Recombinant C1 domains containing the deletion are insoluble and retained intracellularly, indicating that the mutation has detrimental effects on domain folding and structure. Despite this, mutant α2(VI) chains retain the ability to associate into monomers, dimers, and tetramers. However, we show that secreted mutant tetramers containing structurally abnormal C1 VWA domains are unable to associate further into microfibrils, directly demonstrating the critical importance of a correctly folded α2(VI) C1 domain in microfibril formation.     

The C5 domain of the collagen VI alpha3(VI) chain is critical for extracellular microfibril formation and is present in the extracellular matrix of cultured cells

Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha3(VI) N6-C5 chains, tetramers containing truncated alpha3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha3(VI) chain is not essential for microfibril formation.     

Collagen VI, conformation of A-domain arrays and microfibril architecture

Collagen VI is a ubiquitous extracellular matrix protein that assembles into beaded microfibrils that form networks linking cells to the matrix. Collagen VI microfibrils are typically formed from a heterotrimer of the α1, α2, and α3 chains. The α3 chain is distinct as it contains an extended N terminus with up to 10 consecutive von Willebrand factor type A-domains (VWA). Here, we use solution small angle x-ray scattering (SAXS) and single particle analysis EM to determine the nanostructure of nine of these contiguous A-domains. Both techniques reveal a tight C-shape conformation for the A-domains. Furthermore, using biophysical approaches, we demonstrate that the N-terminal region undergoes a conformational change and a proportion forms dimers in the presence of Zn(2+). This is the first indication that divalent cations interact with collagen VI A-domains. A three-dimensional reconstruction of tissue-purified collagen VI microfibrils was generated using EM and single particle image analysis. The reconstruction showed the intricate architecture of the collagen VI globular regions, in particular the highly structurally conserved C-terminal region and variations in the appearance of the N-terminal region. The N-terminal domains project out from the globular beaded region like angled radial spokes. These could potentially provide interactive surfaces for other cell matrix molecules.     

Aberrant Mitochondria in a Bethlem Myopathy Patient with a Homozygous Amino Acid Substitution That Destabilizes the Collagen VI α2(VI) Chain

Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) sit at opposite ends of a clinical spectrum caused by mutations in the extracellular matrix protein collagen VI. Bethlem myopathy is relatively mild, and patients remain ambulant in adulthood while many UCMD patients lose ambulation by their teenage years and require respiratory interventions. Dominant and recessive mutations are found across the entire clinical spectrum; however, recessive Bethlem myopathy is rare, and our understanding of the molecular pathology is limited. We studied a patient with Bethlem myopathy. Electron microscopy of his muscle biopsy revealed abnormal mitochondria. We identified a homozygous COL6A2 p.D871N amino acid substitution in the C-terminal C2 A-domain. Mutant α2(VI) chains are unable to associate with α1(VI) and α3(VI) and are degraded by the proteasomal pathway. Some collagen VI is assembled, albeit more slowly than normal, and is secreted. These molecules contain the minor α2(VI) C2a splice form that has an alternative C terminus that does include the mutation. Collagen VI tetramers containing the α2(VI) C2a chain do not assemble efficiently into microfibrils and there is a severe collagen VI deficiency in the extracellular matrix. We expressed wild-type and mutant α2(VI) C2 domains in mammalian cells and showed that while wild-type C2 domains are efficiently secreted, the mutant p.D871N domain is retained in the cell. These studies shed new light on the protein domains important for intracellular and extracellular collagen VI assembly and emphasize the importance of molecular investigations for families with collagen VI disorders to ensure accurate diagnosis and genetic counseling.     

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.     

Actinidain-hydrolyzed Type I Collagen Reveals a Crucial Amino Acid Sequence in Fibril Formation

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of α1 and α2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the α1 chain, pepsin cleaved between Asp¹⁰³⁵ and Phe¹⁰³⁶, and actinidain between Gly¹⁰³² and Gly¹⁰³³. Thus, the actinidain-hydrolyzed α1 chain is shorter at the C terminus by three residues, Gly¹⁰³³, Phe¹⁰³⁴, and Asp¹⁰³⁵. In the α2 chain, both proteases cleaved between Glu¹⁰³⁰ and Val¹⁰³¹. We demonstrated that a synthetic nonapeptide mimicking the α1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the α1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.     

The supramolecular organization of collagen VI microfibrils

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.     

Structural correlation between collagen VI microfibrils and collagen VI banded aggregates

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.     

Reprint of "Structural correlation between collagen VI microfibrils and collagen VI banded aggregates" [J. Struct. Biol. 154 (2006) 312-326

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.     

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

Supramolecular Organization of the α121-α565 Collagen IV Network

Collagen IV is a family of 6 chains (α1-α6), that form triple-helical protomers that assemble into supramolecular networks. Two distinct networks with chain compositions of α121 and α345 have been established. These oligomerize into separate α121 and α345 networks by a homotypic interaction through their trimeric noncollagenous (NC1) domains, forming α121 and α345 NC1 hexamers, respectively. These are stabilized by novel sulfilimine (SN) cross-links, a covalent cross-link that forms between Met⁹³ and Hyl²¹¹ at the trimer-trimer interface. A third network with a composition of α1256 has been proposed, but its supramolecular organization has not been established. In this study we investigated the supramolecular organization of this network by determining the chain identity of sulfilimine-cross-linked NC1 domains derived from the α1256 NC1 hexamer. High resolution mass spectrometry analyses of peptides revealed that sulfilimine bonds specifically cross-link α1 to α5 and α2 to α6 NC1 domains, thus providing the spatial orientation between interacting α121 and α565 trimers. Using this information, we constructed a three-dimensional homology model in which the α565 trimer shows a good chemical and structural complementarity to the α121 trimer. Our studies provide the first chemical evidence for an α565 protomer and its heterotypic interaction with the α121 protomer. Moreover, our findings, in conjunction with our previous studies, establish that the six collagen IV chains are organized into three canonical protomers α121, α345, and α565 forming three distinct networks: α121, α345, and α121-α565, each of which is stabilized by sulfilimine bonds between their C-terminal NC1 domains.     

Identification of the NC1 Domain of α3 Chain as Critical for α3α4α5 Type IV Collagen Network Assembly

The network organization of type IV collagen consisting of α3, α4, and α5 chains in the glomerular basement membrane (GBM) is speculated to involve interactions of the triple helical and NC1 domain of individual α-chains, but in vivo evidence is lacking. To specifically address the contribution of the NC1 domain in the GBM collagen network organization, we generated a mouse with specific loss of α3NC1 domain while keeping the triple helical α3 chain intact by connecting it to the human α5NC1 domain. The absence of α3NC1 domain leads to the complete loss of the α4 chain. The α3 collagenous domain is incapable of incorporating the α5 chain, resulting in the impaired organization of the α3α4α5 chain-containing network. Although the α5 chain can assemble with the α1, α2, and α6 chains, such assembly is incapable of functionally replacing the α3α4α5 protomer. This novel approach to explore the assembly type IV collagen in vivo offers novel insights in the specific role of the NC1 domain in the assembly and function of GBM during health and disease.     

Orientation of type VI collagen monomers in molecular aggregates

Type VI collagen, prepared from guanidine extracts of human amnion, contains very little monomeric material, the major forms being dimers and tetramers. In order to study the orientation of the molecules in these aggregates, they were digested with pepsin followed by bacterial collagenase. Two fragments were isolated, one containing part of the inner globular domain still attached to part of the triple helix and the other containing large fragments of the outer globular domain. Each fraction was further analyzed; peptides were isolated and their amino-terminal amino acid sequences determined. By comparing the determined sequences with published data, it was found that the outer globular domain contained sequences derived from the amino-terminal domain of all three chains of type VI collagen whereas the inner globular domain contained sequences from the carboxy-terminal domain. This provided direct chemical evidence that dimers and tetramers of type VI collagen are formed by overlapping carboxy-terminal regions of the monomers.     

Hypoxia-inducible Factor-1 (HIF-1) but Not HIF-2 Is Essential for Hypoxic Induction of Collagen Prolyl 4-Hydroxylases in Primary Newborn Mouse Epiphyseal Growth Plate Chondrocytes

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α₂β₂ tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I–III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.     

Structural Insights into Aβ42 Oligomers Using Site-directed Spin Labeling

Oligomerization of the 42-residue peptide Aβ42 plays a key role in the pathogenesis of Alzheimer disease. Despite great academic and medical interest, the structures of these oligomers have not been well characterized. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy is a powerful approach for studying structurally ill-defined systems, but its application in amyloid oligomer structure study has not been systematically explored. Here we report a comprehensive structural study on a toxic Aβ42 oligomer, called globulomer, using site-directed spin labeling complemented by other techniques. Transmission electron microscopy shows that these oligomers are globular structures with diameters of ∼7–8 nm. Circular dichroism shows primarily β-structures. X-ray powder diffraction suggests a highly ordered intrasheet hydrogen-bonding network and a heterogeneous intersheet packing. Residue-level mobility analysis on spin labels introduced at 14 different positions shows a structured state and a disordered state at all labeling sites. Side chain mobility analysis suggests that structural order increases from N- to C-terminal regions. Intermolecular distance measurements at 14 residue positions suggest that C-terminal residues Gly-29–Val-40 form a tightly packed core with intermolecular distances in a narrow range of 11.5–12.5 Å. These intermolecular distances rule out the existence of fibril-like parallel in-register β-structures and strongly suggest an antiparallel β-sheet arrangement in Aβ42 globulomers.     

Kinked collagen VI tetramers and reduced microfibril formation as a result of Bethlem myopathy and introduced triple helical glycine mutations.

Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the dominantly inherited disorder, Bethlem myopathy. Glycine mutations that interrupt the Gly-X-Y repetitive amino acid sequence that forms the characteristic collagen triple helix have been defined in four families; however, the effects of these mutations on collagen VI biosynthesis, assembly, and structure have not been determined. In this study, we examined the consequences of Bethlem myopathy triple helical glycine mutations in the alpha1(VI) and alpha2(VI) chains, as well as engineered alpha3(VI) triple helical glycine mutations. Although the Bethlem myopathy and introduced mutations that are toward the N terminus of the triple helix did not measurably affect collagen VI intracellular monomer, dimer, or tetramer assembly, or secretion, the introduced mutation toward the C terminus of the helix severely impaired association of the mutant alpha3(VI) chain with alpha1(VI) and alpha2(VI). Association of the three chains was not completely prevented, however; and some non-disulfide bonded tetramers were secreted. Examination of the secreted Bethlem myopathy and engineered mutant collagen VI by negative staining electron microscopy revealed the striking finding that in all the cell lines a significant proportion of the tetramers contained a kink in the supercoiled triple helical region. Collagen VI tetramers from all of the mutant cell lines also showed a reduced ability to form microfibrils. These results provide the first evidence of the biosynthetic consequences of collagen VI triple helical glycine mutations and indicate that Bethlem myopathy results not only from the synthesis of reduced amounts of structurally normal protein but also from the presence of mutant collagen VI in the extracellular matrix.     

Covalent and non-covalent interactions of betaig-h3 with collagen VI. Beta ig-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils

Transforming growth factor-beta induced gene-h3 (betaig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that betaig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 x 10(-8) m. Rotary shadowing of immunogold-labeled complexes of recombinant betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.     

A Novel Monoclonal Antibody to Human Laminin α5 Chain Strongly Inhibits Integrin-Mediated Cell Adhesion and Migration on Laminins 511 and 521

Laminins, a large family of αβγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5β1γ1) and 521 (α5β2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3β1/α6β1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3β1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMβ1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.