Collagen is a major component of the extracellular matrix of all mammalian connective tissues. In addition to providing structural support, collagen can also affect cell behavior and gene expression through interactions with other matrix proteins and cellular receptors. We currently recognize 19 genetically distinct collagen types, and numerous other proteins have been described that contain collagenous domains. The collagen triple helix is formed by repeating GXY sequences within each chain, where X is often proline and Y is often hydroxyproline. Such molecules then interact to form higher structures of varying organization, such as fibrils (types I, II,

Identifying mutations that cause specific osteochondrodysplasias will provide novel insights into the function of genes that are essential for skeletal morphogenesis. We report here that an autosomal dominant form of Stickler syndrome, characterized by mild spondyloepiphyseal dysplasia, osteoarthritis, and sensorineural hearing loss, but no eye involvement, is caused by a splice donor site mutation resulting in "in-frame" exon skipping within the COL11A2 gene, encoding the alpha 2(XI) chain of the quantitatively minor fibrillar collagen XI. We also show that an autosomal recessive disorder with similar, but more severe, characteristics is linked to the COL11A2 locus and is caused by a glycine to arginine substitution in alpha 2(XI) collagen. The results suggest that mutations in collagen XI genes are associated with a spectrum of abnormalities in human skeletal development and support the conclusion of others, based on studies of murine chondrodysplasia, that collagen XI is essential for skeletal morphogenesis.

Abstract. It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril. major question in cell biology is how individual macromolecules combine to form the often large and complex supramolecular structures of the extracellular matrix. Approaches to this problem include the direct microscopic visualization of tissue sections aided by chemical stains and immunological tools, reconstitution and binding studies with purified components in solution followed by analysis of the resulting products, and, where possible, direct x-ray analysis of highly ordered arrays in situ.This strategy is particularly well exemplified by the many detailed studies of collagen fibrils and fibrillogenesis. This work has resulted in an understanding of the interactions that govern lateral association of fibrillar collagens (for recent reviews see references 3, 7, and 19). The quarter stagger model that arose from these studies has been considerably refined since its introduction, but still remains as the cornerstone of current models as it explains how fibrils formed in vitro from purified collagen molecules give rise to the characteristic staining patterns observed in the electron microscope. Although the fibril staining patterns produced in vitro match those seen in vivo, the diameter regulation of collagen fibrils in vivo and their complexity, including association with other components, especially proteoglycans, are features not reproduced by mixing solutions of collagens in vitro. In summary, two critical questions remain: how is the construction of fibrils regulated in vivo and what role might other molecules play in these processes?This problem is particularly well illustrated in cartilage. The morphology of fibrils reconstituted from purified type II collagen in solution is vastly different from that of cartilage fibrils in situ. Large tactoidal aggregates with d-periodic staining patterns are formed under appropriate reconstitution conditions (21). In contrast, fibrils from chicken embry...

Clones of embryonic chick chondrocytes have been isolated and collagen biosynthesis has been followed as the clones grow and eventually lose division capacity. Analysis of collagen type at each successive subculture until the time of cellular senescence has shown that a change in synthesis occurs from the cartilage-specific Type II collagen (chain composition Ial(II)H) to a mixture of Type I collagen (chain composition [al(I) MATERIALS AND METHODS Materials. F-10 medium containing twice the usual concentrations of amino acids and pyruvate (F-10 2 X), trypsin (2.5%), bovine-serum albumin (Fraction V), fetal calf serum, Ca++_, and Mg++-free saline, and glutamine were obtained from the Grand Island Biological Co. The radioactive precursor [2-3H]glycine (6.9 Ci/mmol) was obtained from the New England Nuclear Corp. Carrier Type I and Type II chick collagens were prepared as described previously (6). Ascorbic acid, f3-aminopropionitrile fumarate, and BrdUrd were purchased from the Sigma Chemical Co.Cell Culture and Cloning Procedures. Chondrocytes isolated from the sterna of 13-day chick embryos were grown for 4-5 days without feeding in medium F-10 2 X plus 10% fetal calf serum (vol/vol) and 1% bovine-serum albumin (wt/vol) as described previously (7). To isolate clones, the cells selected as "floaters" (4) were first centrifuged from the medium and incubated for 10 min at 370 in Ca++-and Mg++-free saline containing 0.06% trypsin. After centrifugation, cells were resuspended in F-10 2 X and a finely-drawn glass micropipette was used to withdraw a single cell, which was placed within a drop of medium located at the center of a 60 mm tissue culture dish (Falcon Plastics) (4, 8). Several dishes were prepared and each dish was incubated at 370 for 4-6 hr until all cells attached. Medium (3 ml) was added to dishes and the morphology and growth characteristics of each clone were observed daily. Only those clones that grew rapidly and initially possessed the distinct polygonal morphology of chondrocytes were retained. By these criteria, successful clones were obtained from 20-30% of all single cells. Occasionally clones of fibroblasts were observed (5/208 chondrocyte clones) and these were also retained. After about 3 weeks, cells in the chondrocyte clones were enveloped with matrix and some of the cells began to float away from the central mass of chondrocytes and to form secondary colonies. At this time the cells were dissociated with 0.1% trypsin in Ca++-and Mg++-free saline for 45 min. At the first subculture cells were replated into two or more 60 mm tissue culture dishes at 50,000/ml, and at subsequent subcultures were replated into 100 mm dishes at 100,000/ml. When a high cell density was achieved after each subculture, the cells in one or more of the dishes were incubated for 24 hr with [2-3H]glycine (50-100 ,uCi/ml) in the presence of (3-aminopropionitrile (100 ug/ml) and ascorbic acid (50 ,g/ml) in order to label newly-synthesized collagen.Isolation of Collagen from Cell Cultures. The procedures used to i...

Decorin belongs to a family of small leucine-rich proteoglycans that are directly involved in the control of matrix organization and cell growth. Genetic evidence indicates that decorin is required for the proper assembly of collagenous matrices. Here, we sought to establish the precise binding site of decorin on type I collagen. Using rotary shadowing electron microscopy and photoaffinity labeling, we mapped the binding site of decorin protein core to a narrow region near the C terminus of type I collagen. This region is located within the cyanogen bromide peptide fragment ␣1(I) CB6 and is ϳ25 nm from the C terminus, in a zone that coincides with the c 1 band of the collagen fibril D-period. This location is very close to one of the major intermolecular cross-linking sites of collagen heterotrimers. Thus, decorin protein core possesses a unique binding specificity that could potentially regulate collagen fibril stability.

The C-terminal G domains of laminin ␣ chains have been implicated in various cellular and other interactions. The G domain of the ␣4 chain was now produced in transfected mammalian cells as two tandem arrays of LG modules, ␣4LG1-3 and ␣4LG4 -5. The recombinant fragments were shown to fold into globular structures and could be distinguished by specific antibodies. Both fragments were able to bind to heparin, sulfatides, and the microfibrillar fibulin-1 and fibulin-2. They were, however, poor substrates for cell adhesion and had only a low affinity for the ␣-dystroglycan receptor when compared with the G domains of the laminin ␣1 and ␣2 chains. Yet antibodies to ␣4LG1-3 but not to ␣4LG4 -5 clearly inhibited ␣ 6 ␤ 1 integrin-mediated cell adhesion to laminin-8, indicating the participation of ␣4LG1-3 in a cell-adhesive structure of higher complexity. Proteolytic processing within a link region between the ␣4LG3 and ␣4LG4 modules was shown to occur during recombinant production and in endothelial and Schwann cell culture. Cleavage could be attributed to three different peptide bonds and is accompanied by the release of the ␣4LG4 -5 segment. Immunohistology demonstrated abundant staining of ␣4LG1-3 in vessel walls, adipose, and perineural tissue. No significant staining was found for ␣4LG4 -5, indicating their loss from tissues. Immunogold staining demonstrated an association of the ␣4 chain primarily with microfibrillar regions rather than with basement membranes, while laminin ␣2 chains appear primarily associated with various basement membranes.The protein family of laminins consists of at least 12 different isoforms, which are mainly localized in basement membranes. They are involved in major biological functions such as interactions with cellular receptors and the formation of networks that are intermingled with and bound to networks of collagen type IV (1, 2). Most of these heterotrimeric isoforms consist of ␤1/␤2 and ␥1 chains but differ in their ␣ chains, ␣1 to ␣5. The ␣4 chain (200 kDa) is the shortest variant known so far and is present in laminin-8 (␣4␤1␥1) and laminin-9 (␣4␤2␥1) (3-5). The existence of such relatively small laminins was originally indicated from biosynthetic studies with endothelial and adipose cells (6, 7), but their molecular nature was only understood after the complete human (8, 9) and mouse (4, 10, 11) ␣4 chain sequences became available. The domain structure of the ␣4 chain (1816 residues) predicted a small Nterminal region contributing a truncated short arm structure, a coiled-coil domain II/I used for chain association, and a large C-terminal G domain. This prediction was confirmed by electron microscopy of laminin-8 and -9, which lacked one of the three short arm structures found in other laminins (3, 4).Northern and in situ hybridization demonstrated a moderate to strong expression of the ␣4 chain in heart, lung, skeletal muscle, and skin, while some other tissues were negative (4, 8 -11). The chain was also expressed at midgestation stages of mouse development (4, 11) and in va...

We report that mutation of COL11A2 causes deafness previously mapped to the DFNA13 locus on chromosome 6p. We found two families (one American and one Dutch) with autosomal dominant, non-syndromic hearing loss to have mutations in COL11A2 that are predicted to affect the triple-helix domain of the collagen protein. In both families, deafness is non-progressive and predominantly affects middle frequencies. Mice with a targeted disruption of Col11a2 also were shown to have hearing loss. Electron microscopy of the tectorial membrane of these mice revealed loss of organization of the collagen fibrils. Our findings revealed a unique ultrastructural malformation of inner-ear architecture associated with non-syndromic hearing loss, and suggest that tectorial membrane abnormalities may be one aetiology of sensorineural hearing loss primarily affecting the mid-frequencies.

Analysis has been made of the minor collagens which are solubilized by limited pepsin digestion of chicken sterna and which remain in solution after precipitation of type I1 collagen at 0.9 M NaCl-0.5 M HOAc. The precipitate obtained by further dialysis to 1.2 M NaCl-0.5 M HOAc was shown to contain the l a , 2a, and 3a chains previously isolated