Type I collagen was originally reported to contain a single site of 3Hyp. The enzyme complex composed of prolyl 3hydroxylase-1, cartilage associated protein and cyclophilin B was shown to catalyze the 3-hydroxylation of this proline substrate. However, several additional sites have since been reported in A-clade and B-clade collagens, many of which exhibit pronounced enzyme and tissue specificity. This is particularly evident in the C-terminal n motif of type I collagen, where the 3-hydroxylation is unique to tendon and completely Danshensu absent in skin and bone. Differential tissue expression of the three members of the P3H family probably explains this observation. Indeed, recent results have shown that P3H2 expression in tendon is significantly higher than for P3H1 or P3H3. To assess the evolutionary origins of the n domain as a substrate, we surveyed the pa ern of 3Hyp occupancy from early chordates through amphibians, birds and mammals. We examined multiple tissue sources of type I collagen and type II collagen homologs. Type II collagen was included in our study because the known fibrillar collagen genes of pre-vertebrates have sequence features resembling COL2A1. In the current study evolutionary and tissue-specific variations for prolyl 3-hydroxylation were investigated. The present results support a concept that 3-hydroxyproline residues contribute fundamentally to collagen structure and the diversification of connective tissues. Prolyl 3-hydroxylation is a highly conserved collagen pos ranslational modification found throughout the animal kingdom. The evolutionary origins of this ancient modification can be traced as far back as porifera, the most Sipeimine primitive extant multicellular animal. Genomic duplications in early chordates gave rise to the three P3H isoenzymes encoded in higher vertebrate genomes. P3H2 is the predicted modifying enzyme for type IV collagen substrates in the basement membrane, suggesting that P3H2 may be the most conserved in substrate specificity of the duplicated and evolving isoenzymes. It has been demonstrated using cell line RNA interference that the n motif of collagen types I and II is a substrate modified by P3H2. The n motif of fibrillar collagens therefore appears to have evolved as a potential substrate for a pre-existing modifying enzyme that is used to hydroxylate such sequences in type IV collagen. The degree of prolyl 3-hydroxylation may be dependent on the tissue expression pa erns of the enzyme. The n 3Hyp appears to have arisen prior to early vertebrate evolution, as low levels of the modification were detected in lamprey tissues. Low levels of 3Hyp were also found equally distributed across type I collagen from xenopus bone, skin and tendon. Nevertheless, in screening the n motif of type I collagens prepared from different major tissue types, significant 3Hyp occupancy was unique to tendon. Tissue specificity is first observed in chicken, where the 3-hydroxylation is more prevalent in tendon type I collagen. In mammals, 3-hydroxylation of the n in type I collagen 
appears to have become exclusively regulated in tendon. Tendons first evolved as sheet-like structures that transmi ed muscle force over a wide area, such as myosepta in Cephalochordates.