Abnormal collagen metabolism in cultured cells in osteogenesis imperfecta.

Abstract

Haziv, Israel, March 24-28, 1974. biosynthetic studies. To obtain maximal labeling of collagenous protein, the growth media were replaced with 10 ml of the Dulbecco-Vogt medium lacking glycine, lysine, glutamate, and serum but supplemented with ascorbic acid (50 ug/ml) and ,3-aminopropionitrile (50 Ag/ml) (10). Twenty microcuries each of [U-'4C]glycine, [U-14C]proline, and [U-14C]-lysine were added to each flask. Usually cells were exposed to label for 24 hr, except in the studies designed to measure the extractability and synthetic rates of collagen where labeling was carried out for 96 hr without ,3-aminopropionitrile.Preparation of Labeled Proteins. At the termination of the labeling period, phenylmethylsulfonylfluoride (50 Ag/ml) was added to the medium as well as to the cell layer suspension to suppress possible proteolytic alterations of the labeled proteins (11). The cell layer was suspended in 10 ml of 1 M NaCl, 0.05 M Trisc HCl (pH 7.5) at 40 for 1-3 days; insoluble material was recovered by centrifugation. The cell layers from cultures labeled without 0-aminopropionitrile were extracted with 1 M NaCl as above, and the residues were re-extracted with 10 ml of 0.5 M acetic acid and subsequently with 5 ml of 2 M CaCl2.Analysis and Chromatography of Labeled Collagens. After dialysis of medium and cell layer fractions, we estimated the proportion of collagenous and noncollagenous protein by a specific enzymatic method, using bacterial collagenase (12).

Patients with Ehlers-Danlos syndrome type IV lack type III collagen.

“…Sci. USA 72 (1975) We studied cultured collagenous material by a limited cleavage of medium and cell collagenous protein with pepsin to dissolve insoluble collagen and convert precursor forms to collagen (14). Type I and type III chains were separated by standard chromatographic methods (9)(10)(11).…”

“…Skin biopsies were obtained from the left forearm of all other patients with a 4 mm punch. The (12)(13)(14).…”

Reduced secretion of structurally abnormal type I procollagen in a form of osteogenesis imperfecta.

“…In reexamining cultured cells derived from a well-studied patient (6,14,15,17,20) with lethal perinatal 01, we have found that the type I procollagen synthesized by these cells is not secreted at a normal rate, and we suggest that failure of secretion is secondary to a structural mutation in one or both proal(I) chains.…”

“…Affected infants have major abnormalities in bone formation and extremely friable connective tissue (15). The distribution of clinical findings corresponds to the distribution of type I collagen in tissues (16), and decreased production of type I collagen has been observed in cells from a patient with lethal perinatal 01 (6,17).…”

Mutations in collagen genes. Consequences for rare and common diseases.

“…In one of the best studied variants of this type, a deletion of -500 base pairs was produced by a sporatic mutation of one allele for the proal(I) chain (17)(18)(19)(20)(21). The deletion excised three exons with 252 base pairs of coding sequences (21).…”