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...

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2, osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.The small leucine-rich repeat proteoglycans (or SLRPs) 1 are a group of extracellular proteins (ECM) that belong to the leucine-rich repeat (LRR) superfamily of proteins (1, 2). The LRR is a protein folding motif composed of 20 -30 amino acids with leucines in conserved positions. LRR-containing proteins are present in a broad spectrum of organisms and possess diverse cellular functions and localization (3). The members of the SLRP subfamily have core proteins of similar size (about 40 kilodaltons) that are dominated by a central domain composed of 6 -10 tandemly repeated LRRs. This domain is flanked by smaller, less conserved N-terminal and C-terminal regions containing cysteines in characteristic positions.Most of the SLRP proteins are proteoglycans, and the SLRP gene family has been subdivided into 3 classes based on similarities in overall amino acid sequence, spacing of cysteine residues in the N terminus, and gene structure. The previously identified class I members, decorin (4) and biglycan (5), are the most closely related SLRPs based on amino acid sequences; the human sequences are 57% identical. The core proteins contain 10 LRRs, and the N-terminal regions of decorin and biglycan are substituted with one and two chondroitin/dermatan sulfate chains, respectively. The cysteine-rich cluster in the N terminus of class I SLRPs has an amino acid spacing of CX 3 CXCX 6 C. The mouse decorin (6) and biglycan genes (7) contain 8 exons.The class II members, fibromodulin (8), lumican (9), PRELP (10), keratocan (11), and osteoadherin (12), have a pairwise amino acid sequence identity between 37 and 55% and have a common gene structure composed of three exons. The cysteine spacing in the N-terminal region of class II SLRPs is identical (CX 3 CXCX 9 C) but different from the other SLRP classes. The core pro...

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