Evolutionary Origins of C-Terminal (GPP)n 3-Hydroxyproline Formation in Vertebrate Tendon Collagen

Abstract

Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

“…4B). Type I collagen also has additional tendon-specific prolyl 3-hydroxylation sites in the C-terminal (GPP) n sequence of the triple helix (30,31). From the present findings it is clear that as in tendon, scleral type I collagen from wild-type mice exhibits this characteristic "hydroxylation ladder" (Fig.…”

“…Protein sequences used for MS analysis were obtained from the Ensembl genome database. Mass spectral quantitation of 3Hyp residues was based on 16-Da mass increases at known sites of prolyl 3-hydroxylation at the Xaa position of Gly-Xaa-4Hyp sequences previously confirmed by Edman degradation (5,28,30,31).…”

“…The percentages were determined based on the ratio the m/z peaks of each post-translational variant as described previously (31). No notable differences were observed in type I collagen 3Hyp occupancy between mouse sclera and cornea (cornea not shown).…”

Ancient proteins resolve the evolutionary history of Darwin’s South American ungulates

“…We identified several regions, mainly LETTER RESEARCH located in COL1a1, that appeared to lack sequence variation among the four major mammalian superorders. This could be a result of the functional importance of some of these regions during COL1 fibril formation, a1 and a2 chain binding, and COL1 hydroxylation [36][37][38] . Additionally, we observed a substitution rate in COL1a2 roughly twice that observed in COL1a1.…”

P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA

“…This was surprising, particularly for P3h3 Ϫ/Ϫ , which is a close homolog of P3h1 and P3h2, including a potentially active catalytic domain. Both P3h1 and P3h2 have evolved preferred GPP substrate sequences in a range of collagen types (17,24,25).…”