Studies on the molecular composition and degradation of type IV procollagen.

The effects of trypsin on soluble type IV procollagen from the EHS mouse tumour were studied. The enzyme cleaved the pro alpha 1(IV) and pro alpha 2(IV) chains, causing only a minor decrease in the molecular weight of the pro alpha 1(IV) chain, whereas the pro alpha 2(IV) chain was degraded to at least two smaller peptides. Analyses of the uncleaved and trypsin-digested type IV procollagen by molecular sieving, with and without reduction and denaturation, were consistent with the two chains, pro alpha 1(IV) and pro alpha 2(IV), being in the same molecule, as a heterotrimer with the composition [pro alpha 1(IV)]2pro alpha 2(IV).

Cortisol decreases the cellular concentration of translatable procollagen mRNA species in cultured human skin fibroblasts.

The effect of cortisol on the cellular concentration of translatable procollagen mRNAs was studied in cultured human skin fibroblasts. Cortisol selectively decreased the amount of procollagen mRNAs, in comparison to the total mRNA activity, when the cells were grown in enriched medium conditions, i.e.

Protein disulphide-isomerase activity in various cells synthesizing collagen.

A high correlation was found between the activities of protein disulphide isomerase and prolyl 4-hydroxylase when assayed in cells synthesizing various collagen types or the same type at markedly different rates. The highest activities of both enzymes were found in freshly isolated chick-embryo tendon and cartilage cells, intermediate activities in confluent cultures of human skin and lung fibroblasts and mouse 3T6 fibroblasts, and the lowest values in three human sarcoma cell lines, the difference in protein disulphide isomerase activity between the freshly isolated tendon cells and confluent simian-virus-40-transformed human lung fibroblasts being about 25-fold. All these differences are in good agreement with differences reported between the various cells in their rates of collagen synthesis.

Synthesis of a shortened pro-alpha 2(I) chain and decreased synthesis of pro-alpha 2(I) chains in a proband with osteogenesis imperfecta.

Synthesis of type I procollagen was examined in skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta. The fibroblasts synthesized shortened pro-alpha 2(I) chains and these shortened chains accounted for all the pro-alpha 2(I) chains synthesized by the cells. In addition, there was a decrease in the relative rate of synthesis of pro-alpha 2(I) chains.

Effects of streptozotocin diabetes, glucose, and insulin on the metabolism of type IV collagen and proteoglycan in murine basement membrane-forming EHS tumor tissue.

The influence of streptozotocin diabetes, glucose, and insulin on the metabolism of basement membrane collagen and proteoglycan was studied in the murine EHS tumor. No differences were found in the amino acid composition of tumors grown in diabetic and control mice, suggesting that the relative amount of collagen was not increased. Similarly, no significant differences were observed in any of the intracellular enzyme activities of collagen biosynthesis in the tumor tissue, but in diabetic mice, a significant increase was found in all four enzyme activities studied in the kidneys.

Posttranslational modifications in the biosynthesis of type IV collagen by a human tumor cell line.

Factors responsible for the high extent of intracellular posttranslational modifications in type IV collagens were studied in a cultured human tumor cell line, HT-1080. These cells do not synthesize any detectable amounts of interstitial collagens but produce type IV collagen at a high rate, corresponding to about one-third of the production of interstitial collagens by cultured human skin fibroblasts. Prolyl 4-hydroxylase activity was lower in the HT-1080 cells than in human skin fibroblasts, there being a rough correlation between this enzyme activity and the rate of 4-hydroxyproline formation in these two cell types.

Differences between proline and lysine hydroxylations in their inhibition by zinc or by ascorbate deficiency during collagen synthesis in various cell types.

The addition of Zn2+ inhibited lysine hydroxylation markedly less effectively than it did proline hydroxylation in chick embryo tendon cells, 3T6 fibroblasts and lysyl hydroxylase-deficient Ehlers-Danlos Syndrome Type VI fibroblasts. With low Zn2+ concentrations, a similar difference was also seen in chick embryo cartilage cells, whereas with high concentrations both hydroxylations were affected to the same extent in this cell type. Ascorbate deficiency likewise had a much less effect on lysine than proline hydroxylation when studied with 3T6 fibroblasts.

Deficiency of galactosylhydroxylysyl glucosyltransferase, an enzyme of collagen synthesis, in a family with dominant epidermolysis bullosa simplex.

Members of a family with dominant epidermolysis bullosa simplex were found to have a deficiency of galactosylhydroxylysyl glucosyltransferase (GGT), an enzyme catalyzing the glucosylation of galactosylhydroxylysyl residues in the biosynthesis of collagen. The enzyme's activity was low in serum, skin tissue, and cultured skin fibroblasts, although no abnormality was found in three other intracellular enzymes of collagen biosynthesis. Mixtures of serum samples from patients and healthy controls gave the expected GGT activity, indicating that the low values were not due to inhibitors.