SMALL MOLECULAR WEIGHT HYDROXYPROLINE-CONTAINING COMPOUNDS IN THE DEVELOPING MOUSE LIMB

Abstract

We have found 3 small molecular weight hydroxyproline (OHPro)-containing compounds that are abundant in the developing mouse limb. The amino acid OHPro is customarily related to the presence of collagen and elastin, molecules in the extracellular matrix that contain OHPro. Cells cannot incorporate free OHPro directly into collagen during synthesis; instead OHPro is formed by the post-translational hydroxylation of polypeptide-bound proline. In fibroblast cultures (J. Cell Sci. 12:217, 1973) most of the labeled OHPro made from proline is a free amino acid or in a small polypeptide.We have cultured developing limbs from day 10 to 14 embryos with C14-proline for 4 hrs. Snail and large molecular weight fractions were obtained by chromatography on Sephadex G-25. The amount of C14-OHPro was determined on an amino acid analyzer column. Over 80% of the C14-OHPro was in the small molecular weight fractions and included trans-4-OHPro, Cis-4-OHPro and trans-3-OHPro. These compounds were also produced in the presence of “,αα-dipyridyl, which inhibits the known hydroxylation enzyme. The presence of Cis-4-OHPro is a surprise as it prevents the formation of a stable collagen helix. This compound has never been isolated from mammalian tissues. Trans-3-OHPro is abundant in type TV collagen, the most common source of which is basement membranes. The role (if any) of these 3 srall molecular weight OH-Pro compounds in development remains to be elucidated. Supported by NIH Grants 1009689 and HL18714.