We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the ␣4(VI), ␣5(VI), and ␣6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (␣4 and ␣6 chains) or three (␣5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the ␣6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse ␣4(VI)N6-C2 chain co-assembled with endogenous ␣1(VI) and ␣2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, ␣5(VI)N5-C1 and ␣6(VI)N6-C2 chains did not assemble with ␣1(VI) and ␣2(VI) chains and accumulated intracellularly. We conclude that the ␣4(VI)N6-C2 chain contains all the elements necessary for trimerization with ␣1(VI) and ␣2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.Collagen VI is an extracellular component that is present in virtually all connective tissues, where it forms abundant and structurally unique microfibrils in close association with basement membranes. Collagen VI interacts with a range of ECM 2 components. However, its precise role is not clearly understood. Several recent studies have suggested that collagen VI functions to anchor the basement membrane to the pericellular matrix in muscle (1-3). Other data suggest a role for collagen VI in cell signaling and cell migration (4, 5).Three genetically distinct collagen VI chains, ␣1(VI), ␣2(VI), and ␣3(VI), encoded by the COL6A1, COL6A2, and COL6A3 genes were first described more than 20 years ago (6 -8). The COL6A1 and COL6A2 genes are located in tandem on chromosome 21q22.3. The ␣1(VI) and ␣2(VI) chains are similar in size and domain structure. They contain a short 335-or 336-amino acid triple helix with a glycine triplet repeat motif that is characteristic of all collagens. Flanking the triple helix are domains homologous to the A-type domains found in von Willebrand factor (VWA domains). ␣1(VI) and ␣2(VI) contain one VWA domain N-terminal to the triple helix (N1) and two VWA domains on the C-terminal flank of the helix (C1 and C2). In contrast, the ␣3(VI) chain, encoded by the COL6A3 gene on 2q37.3, is much larger with 10 N-terminal (N1-N10), two C-terminal VWA domains (C1 and C2), and several other identifiable types of domains at the C terminus (C3-C5).A...

Here we describe a novel specific component of tissue junctions, collagen XXII. It was first identified by screening an EST data base and subsequently expressed as a recombinant protein and characterized as an authentic tissue component. The COL22A1 gene on human chromosome 8q24.2 encodes a collagen that structurally belongs to the FACIT protein family (fibril-associated collagens with interrupted triple helices). Collagen XXII exhibits a striking restricted localization at tissue junctions such as the myotendinous junction in skeletal and heart muscle, the articular cartilage-synovial fluid junction, or the border between the anagen hair follicle and the dermis in the skin. It is deposited in the basement membrane zone of the myotendinous junction and the hair follicle and associated with the extrafibrillar matrix in cartilage. In situ hybridization of myotendinous junctions revealed that muscle cells produce collagen XXII, and functional tests demonstrated that collagen XXII acts as a cell adhesion ligand for skin epithelial cells and fibroblasts. This novel gene product, collagen XXII, is the first specific extracellular matrix protein present only at tissue junctions.

Decorin is a multifunctional small leucine‐rich proteoglycan involved in the regulation of collagen fibrillogenesis. In patients with a variant of Ehlers–Danlos syndrome, about half of the secreted decorin lacks the single glycosaminoglycan side chain. Notably, these patients have a skin‐fragility phenotype that resembles that of decorin null mice. In this study, we investigated the role of glycanated and unglycanated decorin on collagen fibrillogenesis. Glycosaminoglycan‐free decorin, generated by mutating Ser4 of the mature protein core into Ala (DCN‐S4A), showed reduced inhibition of fibrillogenesis compared with the decorin proteoglycan. Interestingly, using a 3D matrix generated by decorin‐null fibroblasts, an increase in fibril diameter was found after the addition of decorin, and even greater effects were observed with DCN‐S4A. To avoid potential side effects of artificial tags, adenoviruses containing decorin and DCN‐S4A were used to transduce decorin‐null fibroblasts prior to matrix formation. Both molecules were efficiently incorporated into the matrix, with no changes in collagen composition and network formation, or altered expression of the related proteoglycan biglycan. Both decorin and DCN‐S4A mutants increased the collagen fibril diameter, with the latter showing the most prominent effects. These data show that at early stages of fibrillogenesis, the glycosaminoglycan chain of decorin has a reducing effect on collagen fibril diameter.

All morphogens of the Hedgehog (Hh) family are synthesized as dual-lipidated proteins, which results in their firm attachment to the surface of the cell in which they were produced. Thus, Hh release into the extracellular space requires accessory protein activities. We suggested previously that the proteolytic removal of N-and Cterminal lipidated peptides (shedding) could be one such activity. More recently, the secreted glycoprotein Scube2 (signal peptide, cubulin domain, epidermal-growth-factor-like protein 2) was also implicated in the release of Shh from the cell membrane. This activity strictly depended on the CUB domains of Scube2, which derive their name from the complement serine proteases and from bone morphogenetic protein-1/tolloid metalloproteinases (C1r/C1s, Uegf and Bmp1). CUB domains function as regulators of proteolytic activity in these proteins. This suggested that sheddases and Scube2 might cooperate in Shh release. Here, we confirm that sheddases and Scube2 act cooperatively to increase the pool of soluble bioactive Shh, and that Scube2-dependent morphogen release is unequivocally linked to the proteolytic processing of lipidated Shh termini, resulting in truncated soluble Shh. Thus, Scube2 proteins act as protease enhancers in this setting, revealing newly identified Scube2 functions in Hh signaling regulation.

Age-related macular degeneration (ARMD) with abnormal deposit formation under the retinal pigment epithelium (RPE) is the major cause of blindness in the Western world. basal laminar deposits are found in early ARMD and are composed of excess basement membrane material produced by the RPE. Here, we demonstrate that mice lacking the basement membrane component collagen XVIII/endostatin have massive accumulation of sub-RPE deposits with striking similarities to basal laminar deposits, abnormal RPE, and age-dependent loss of vision. The progressive attenuation of visual function results from decreased retinal rhodopsin content as a consequence of abnormal vitamin A metabolism in the RPE. In addition, aged mutant mice show photoreceptor abnormalities and increased expression of glial fibrillary acidic protein in the neural retina. Our data demonstrate that collagen XVIII/ endostatin is essential for RPE function, and suggest an important role of this collagen in Bruch's membrane. Consistent with such a role, the ultrastructural organization of collagen XVIII/endostatin in basement membranes, including Bruch's membrane, shows that it is part of basement membrane molecular networks.