Location of 3-Hydroxyproline Residues in Collagen Types I, II, III, and V/XI Implies a Role in Fibril Supramolecular Assembly

Abstract

Collagen triple helices are stabilized by 4-hydroxyproline residues. No function is known for the much less common 3-hydroxyproline (3Hyp), although genetic defects inhibiting its formation cause recessive osteogenesis imperfecta. To help understand the pathogenesis, we used mass spectrometry to identify the sites and local sequence motifs of 3Hyp residues in fibril-forming collagens from normal human and bovine tissues. The results confirm a single, essentially fully occupied 3Hyp site (A1) at Pro 986 in A-clade chains ␣1(I), ␣1(II), and ␣2(V). Two partially modified sites (A2 and A3) were found at Pro 944 in ␣1(II) and ␣2(V) and Pro 707 in ␣2(I) and ␣2(V), which differed from A1 in sequence motif. Significantly, the distance between sites 2 and 3, 237 residues, is close to the collagen D-period (234 residues). A search for additional D-periodic 3Hyp sites revealed a fourth site (A4) at Pro 470 in ␣2(V), 237 residues N-terminal to site 3. In contrast, human and bovine type III collagen contained no 3Hyp at any site, despite a candidate proline residue and recognizable A1 sequence motif. A conserved histidine in mammalian ␣1(III) at A1 may have prevented 3-hydroxylation because this site in chicken type III was fully hydroxylated, and tyrosine replaced histidine. All three B-clade type V/XI collagen chains revealed the same three sites of 3Hyp but at different loci and sequence contexts from those in A-clade collagen chains. Two of these B-clade sites were spaced apart by 231 residues. From these and other observations we propose a fundamental role for 3Hyp residues in the ordered self-assembly of collagen supramolecular structures.Collagens are the most abundant and ubiquitous proteins in multi-cellular animals. It is well established that 4-hydroxyproline (4Hyp) 2 residues stabilize the collagen triple helix through water-bridged intramolecular hydrogen bonding (1). However, the function of the much less abundant 3-hydroxyproline (3Hyp), although discovered 50 years ago, is unknown (2). Only 1-2 residues of 3Hyp occur per chain in collagen types I and II and 3-6 residues occur per chain in collagen types V and XI. The content is highest in type IV collagens of basement membranes in which 10% of the total hydroxyproline can be 3Hyp (3).Specific prolyl 3-hydroxylases (P3Hs) are responsible for 3Hyp synthesis. Three different genes encoding P3H1, P3H2, and P3H3 are present in the human genome, which show tissue specificity in their expression (4, 5). Substrate proline residues occur in a prerequisite sequence -Pro-4Hyp-Gly. The ␣1(I) chain has only one established 3Hyp site at Pro 986 in a motif conserved across vertebrate species (human GLPGPIGPPGPR) a close variant of which also occurs in type II collagen (human GIPGPIGPPGPR).Renewed interest in 3Hyp was recently sparked by the discovery that a recessive form of osteogenesis imperfecta (OI) is caused by mutations in CRTAP. This gene encodes a protein (cartilage-associated protein) that is bound to P3H1 and cyclophilin B in the endoplasmic reticulum and is requi...

Recombinant expression of hydroxylated human collagen in Escherichia coli

“…Hyp occurs exclusively at position y (Fietzek and Rauterberg 1975), recent studies showed that Hyp also occurs at position x in fibrillar collagens (Song and Mechref 2013;Weis et al 2010).…”

Osteogenesis imperfecta

“…During this phase, 65 kDa FK506 binding protein (FKBP65; encoded by FKBP10) and a complex formed by P3H1 -CRTAPPPIase B (peptidyl prolyl cis-trans isomerase B) -seem to play a crucial part. The complex is involved in the hydroxyl ation of proline 986 of the collagen α1(I) chain and α1(II) chain and proline 707 of the α2(I) chain 13,[19][20][21] , which are thought to be important for supramolecular assembly of collagen fibrils and to serve as binding sites for chaperones or small leucine rich proteoglycans 22 . Beyond its prolyl 3 hydroxylase activity, the com plex functions as a PPIase and chaperone for collagen folding 13,19,23 .…”

Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

“…Red, hydroxylated, non-glycosylated residues; green, Glc-Gal-Hyl residues; dark blue, Gal-Hyl residue; purple, residue found as both Glc-Gal-Hyl and Gal-Hyl. Underlined, hydroxylated/glycosylated residues mapped in previous studies (4,32). Sequences not identified in the course of MS analysis are in light blue.…”

“…Recent analyses of collagenous proteins by MS relied heavily on a priori biological knowledge to assess prolyl hydroxylation (i.e. PTM assignment based upon hydroxylation motifs) (30,32) rather than PTM assignment based upon localizing fragments from MS n spectra. We report comprehensive mapping of all PTMs involving hydroxylated residues on bovine placenta ␣1(V) and human recombinant pro-␣1(V) collagen chains and provide manually verified mass spectral evidence for each modified site.…”