Abstract. The aim of this investigation was to identify the domains of type IV collagen participating in cell binding and the cell surface receptor involved. A major cell binding site was found in the trimeric cyanogen bromide-derived fragment CB3, located 100 nm away from the NH: terminus of the molecule, in which the triple-helical conformation is stabilized by interchain disulfide bridges. Cell attachment assays with type IV collagen and CB3 revealed comparable cell binding activities. Antibodies against CB3 inhibited attachment on fragment CB3 completely and on type IV collagen to 80%. The ability to bind cells was strictly conformation dependent.Four trypsin derived fragments of CB3 allowed a closer investigation of the binding site. The smallest, fully active triple-helical fragment was (150)3-amino acid residues long. It contained segments of 27 and 37 residues, respectively, at the NHe and COOH terminus, which proved to be essential for cell binding. By affinity chromatography on Sepharose-immobilized CB3, two receptor molecules of the integrin family, otl/31 and oe2/$1, were isolated. Their subunits were identified by sequencing the NH2 termini or by immunoblotting. The availability of fragment CB3 will allow for a more in-depth study of the molecular interaction of a short, well defined triple-helical ligand with collagen receptors o¢1/~1 and o~2B1.

A minigene version of the human gene for type H procollagen (COI2A1) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened proa chains that associate with normal proa chains and thereby cause degradation of the shortened and normal proa chains through a process called procollagen suicide. Here we have prepared transgenic mice expressing a minigene version of the human COL2AJ gene. The design of the gene construct was based on several mutations in the genes for type I procollagen that produced in-frame deletions in codons for the repeating -Gly-Xaa-Yaa-amino acid sequence of the collagen triple helix and caused lethal variants of 01 (for reviews, see refs. 9 and 10). Because the collagen triple helix is formed by a series of hydrogen bonds and water bridges that link the -Gly-Xaa-Yaa-sequences in one proa chain to equivalent -Gly-Xaa-Yaa-sequences in the two other proa chains (see ref. 13), the presence of one shortened proa chain in a procollagen molecule can prevent folding into a stable triple helix and degradation of all three proa chains in a process referred to as "procollagen suicide" (11, 14, 15). MATERIALS AND METHODSGene Construct. The gene construct was prepared from a cosmid clone containing the human COL2AJ gene (ref. 16; kindly provided by Francesco Ramirez, Brookdale Center for Molecular Biology, Mt. Sinai Medical Center, New York). The insert in the cosmid was cleaved with Xba I, Sph I, and Cla I to generate four fragments ranging in size from 5 to 12 kilobases (kb) (Fig. 1). Three of the fragments were then assembled into a modified cosmid vector (17, 18) by four-way ligation. The insert was digested with Sal I, electrophoresed in an agarose gel, electroeluted, extracted with phenol/ chloroform/isoamyl alcohol (24:24:1), ethanol precipitated, and dissolved in 1 mM EDTA in 10 mM Tris HCl buffer (pH 7.4) for microinjection.Preparation of Transgenic Mice. One-cell stage mouse embryos (19) were obtained by mating of inbred FVB/N males and females. The partially deleted COL2AJ gene was microinjected into embryos at a concentration of 2 ,ug/ml with about 600 copies per embryo. Inbred CD1 females were used as the pseudopregnant recipients.Assay *To whom reprint requests should be addressed. 7640The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The human COL11A2 gene was analyzed from two overlapping cosmid clones that were previously isolated in the course of searching the human major histocompatibility region (Janatipour, M., Naumov, Y., Ando, A., Sugimura, K., Okamoto, N., Tsuji, K., Abe, K., and Inoko, H. (1992) Immunogenetics 35, 272-278). Nucleotide sequencing defined over 28,000 base pairs of the gene. It was shown to contain 66 exons. As with most genes for fibrillar collagens, the first intron was among the largest, and the introns at the 5-end of the gene were in general larger than the introns at the 3-end. Analysis of the exons coding for the major triple helical domain indicated that the gene structure had not evolved with the genes for the major fibrillar collagens in that there were marked differences in the number of exons, the exon sizes, and codon usage. The gene was located close to the gene for the retinoic X receptor ␤ in a head-to-tail arrangement similar to that previously seen with the two mouse genes (P. Vandenberg and D. J. Prockop, submitted for publication). Also, there was marked interspecies homology in the intergenic sequences. The amino acid sequences and the pattern of charged amino acids in the major triple helix of the ␣2(XI) chain suggested that the chain can be incorporated into the same molecule as ␣1(XI) and ␣1(V) chains but not into the same molecule as the ␣3(XI)/␣1(II) chain. The structure of the carboxyl-terminal propeptide was similar to the carboxyl-terminal propeptides of the pro␣1(XI) chain and pro␣ chains of other fibrillar collagens, but it was shorter because of internal deletions of about 30 amino acids.Over 19 types of collagens are known, each with an apparently unique biological function (1-3). A major subclass is the fibrillar collagens that form ordered extracellular fibrils and that include type I, type II, type III, type V, and type XI collagens. Type I and type III collagens are found in most non-cartilaginous tissues. Type II is found primarily in cartilage where it is the most abundant protein, but it is also present in the vitreous humor and several other tissues in early embryonic development. Type XI collagen was originally recognized as a minor fibrillar collagen in cartilage that was similar to type II collagen. The protein was considered to consist of three ␣ chains referred to as ␣1(XI), ␣2(XI), and ␣3(XI) (4 -6). The ␣3(XI) chain was subsequently shown to be derived from the same gene as the ␣1(II) chain of type II collagen that, by an unknown mechanism, was assembled with the ␣1(XI) and ␣2(XI) chain to form a unique procollagen molecule (4 -9). In further analyses, type XI collagen was found to be closely related in structure to type V collagen, and both type V and type XI collagens were found in small amounts in a variety of cartilaginous and non-cartilaginous connective tissues (10, 11). Amino acid sequencing of fragments of collagen fibrils from mammalian vitreous humor demonstrated that fibrils were assembled from molecules containing ␣1(XI) and ␣2(V) chains (12). Also, during th...

Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. NatL. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that -15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d-and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis. (J. Clin. Invest. 1993. 92:582-595.)