Type VI collagen filaments are found associated with interstitial collagen fibers, around cells, and in contact with endothelial basement membranes. To identify type VI collagen binding proteins, the amino-terminal domains of the ␣1(VI) and ␣2(VI) chains and a part of the carboxyl-terminal domain of the ␣3(VI) chain were used as bait in a yeast two-hybrid system to screen a human placenta library. Eight persistently positive clones were identified, two coding the known matrix proteins fibronectin and basement membrane type IV collagen and the rest coding new proteins. The amino-terminal domain of ␣1(VI) was shown to interact with the carboxylterminal globular domain of type IV collagen. The specificity of this interaction was further studied using the yeast two-hybrid system in a one-on-one format and confirmed by using isolated protein domains in immunoprecipitation, affinity blots, and enzyme-linked immunosorbent assay-based binding studies. Co-distribution of type VI and type IV collagens in human muscle was demonstrated using double labeling immunofluorescent microscopy and immunoelectron microscopy. The strong interaction of type VI collagen filaments with basement membrane collagen provided a possible molecular pathogenesis for the heritable disorder Bethlem myopathy.Type VI collagen filaments are ubiquitous. They are present in all connective tissues that contain type I and type III collagen fibers and in cartilage, a tissue that contains predominantly type II collagen. The major functions that have been suggested for type VI collagen filamentous networks are as a substrate for cell attachment and as an anchoring meshwork that connects collagen fibers, nerves, and blood vessels to the surrounding matrix (1, 2). This implies that not only is there an interaction, either direct or indirect, with the type I/III collagen fibers but also that there is an interaction with components of endothelial basement membranes.Matrix components that have been shown to interact with type VI collagen in vitro include proteoglycans, collagens, hyaluronan, heparin, and integrins.Proteoglycans appear to be particularly important, since cell surface-, basement membrane-, and collagen fibril-associated proteoglycans have all been reported to bind to various forms of type VI collagen. Decorin is a small dermatan sulfate proteoglycan that binds to fibrillar collagens (3). It was shown that the leucine-rich module of the core protein bound to type VI and that the binding could be inhibited by the core proteins of fibromodulin and biglycan (4). The cell surface-associated membrane chondroitin sulfate proteoglycan NG2 was originally detected in cells from the rodent central nervous system but subsequently was also detected in blood vessels and cartilaginous structures of the head, neck, and spine. It interacts via its core protein with type VI collagen and is thought to provide machinery for transmembrane signaling (5). Since ␣1␤1 and ␣2␤1 integrins also bind type VI collagen (6, 7), the cell signaling potential of this molecule woul...

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.

Human collagen alpha 3(VI) chain mRNA (approximately 10 kb) was cloned and shown by sequence analysis to encode a 25 residue signal peptide, a large N‐terminal globule (1804 residues), a central triple helical segment (336 residues) and a C‐terminal globule (803 residues). Some of the sequence was confirmed by Edman degradation of peptides. The N‐terminal globular segment consists of nine consecutive 200 residue repeats (N1 to N9) showing internal homology and also significant identity (17‐25%) to the A domains of von Willebrand Factor and similar domains present in some other proteins. Deletions were found in the N3 and N9 domains of several cDNA clones suggesting variation of these structures by alternative splicing. The C‐terminal globule starts immediately after the triple helical segment with two domains C1 (184 residues) and C2 (248 residues) being similar to the N domains. They are followed by a proline rich, repetitive segment C3 of 122 residues, with similarity to some salivary proteins, and domain C4 (89 residues), which is similar to the type III repeats present in fibronectin and tenascin. The most C‐terminal domain C5 (70 residues) shows 40‐50% identity to a variety of serine protease inhibitors of the Kunitz type. The whole sequence contains 29 cysteines which are mainly clustered in short segments connecting domains N1, C1, C2 and the triple helix, and in the inhibitor domain. Five putative Arg‐Gly‐Asp cell‐binding sequences are exclusively localized in the triple helical segment.(ABSTRACT TRUNCATED AT 250 WORDS)

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.