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Biochemical characterization of the native tissue form of type X collagen from embryonic chick sternal cartilage and identification of a chymotrypsin-sensitive site within its triple-helical domain.

ABSTRACT: We isolated and characterized the intact native tissue form of type X collagen from the presumptive calcification region of lathyritic chick-embryo sterna and from organ cultures incubated in the presence of beta-aminopropionitrile (beta APN). The administration of beta-APN in vivo greatly increased the solubility of type X collagen and allowed the extraction of quantitative amounts of these molecules under non-denaturing non-proteolytic conditions. Biosynthetic studies in vitro showed that the addition of beta APN during labelling resulted in a 4-fold increase in the extractability of the newly synthesized type X collagen. Biochemical characterization of the intact type X collagen extracted from the tissues or biosynthesized in the organ cultures showed that type X collagen is composed of 59,000-Mr chains that do not undergo conversion into shorter polypeptides. Despite the marked solubilization of type X collagen upon administration of beta APN, a substantial proportion remained tissue-bound and could only be extracted by employing proteolytic digestion followed by disulphide bond reduction. These findings indicate that type X collagen in the tissues is stabilized by at least two different mechanisms, one involving beta APN-sensitive cross-links and the second through interactions with disulphide-bonded proteins. Limited proteolytic digestion with chymotrypsin of tissues containing 1.0 M-NaCl-insoluble type X collagen resulted in its complete solubilization. The majority of type X collagen molecules extracted with chymotrypsin were approx. 10% shorter than those obtained after limited pepsin digestion (Mr 40,000 versus Mr 45,000) and showed the selective loss of a single CNBr-cleavage peptide. These findings indicate the existence of chymotrypsin-sensitive sites within the triple-helical domain of the molecules.

SUBMITTER: Reginato AM

PROVIDER: S-EPMC1149850 | biostudies-other | 1991 Jan

REPOSITORIES: biostudies-other

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Project description:We have performed a quantitative analysis of the various collagens biosynthesized by organ cultures of whole embryonic-chick sternum and its separate anatomical regions corresponding to the zones of permanent hyaline and presumptive-calcification cartilages. Our studies demonstrated that embryonic-chick sternum devotes a large portion of its biosynthetic commitment towards production of Type X collagen, which represented approx. 18% of the total newly synthesized collagen. Comparison of the collagens biosynthesized by the permanent hyaline cartilage and by the cartilage from the presumptive-calcification zone demonstrated that Type X-collagen production was strictly confined to the presumptive-calcification region. Sequential extraction of the newly synthesized Type X collagen demonstrated the existence of two separate populations. One population (approx. 20%) was composed of easily extractable molecules that were solubilized with 1.0 m-NaCl/50 mM-Tris/HCI buffer, pH 7.4. The second population was composed of molecules that were not extractable even after repeated pepsin digestion, but became completely solubilized after treatment with 20 mM-dithiothreitol/0.15 M-NaCl buffer at neutral pH. These results suggest that most of the Type X collagen normally exists in the tissue as part of a pepsin-resistant molecular aggregate that may be stabilized by disulphide bonds. Quantitative analysis of the proportion of Type X collagen relative to the other collagens synthesized in the cultures indicated that this collagen was a major biosynthetic product of the presumptive-calcification cartilage, since it represented about 35% of the total collagen synthesized by this tissue. In contrast, the permanent hyaline cartilage did not display any detectable synthesis of Type X collagen. When compared on a per-cell basis, the chondrocytes from the presumptive-calcification zone synthesized approx. 33% more Type X collagen than the amount of Type II collagen synthesized by the chondrocytes from the permanent-hyaline-cartilage zone. Subsequently, it was demonstrated that Type X collagen is a structural component of chick sternum matrix, since quantitative amounts could be extracted from the region of presumptive calcification of 17-day-old chick-embryo sterna and from the calcified portion of adult-chick sterna. The strict topographic distribution in the expression of Type X collagen biosynthesis to the zone of presumptive calcification suggests that this collagen may play an important role in initiation or progression of tissue calcification.

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Biochemical characterization of the native tissue form of type X collagen from embryonic chick sternal cartilage and identification of a chymotrypsin-sensitive site within its triple-helical domain.

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