Irina Perdivara’s research while affiliated with Fujifilm Diosynth Biotechnologies and other places

Fibrillar type I collagen is the most abundant structural protein in most tissues and organs. One of the unique and functionally important characteristics of collagen is sequential posttranslational modifications of lysine (Lys) residues. In the endoplasmic reticulum, hydroxylation of specific Lys occurs producing 5-hydroxylysine (Hyl). Then, to the 5-hydroxyl group of Hyl, a single galactose unit can be attached to form galactosyl-Hyl (Gal-Hyl) and further glucose can be added to Gal-Hyl to form glucosylgalactosyl-Hyl (GlcGal-Hyl). These are the only two O-linked glycosides found in mature type I collagen. It has been shown that this modification is critically involved in a number of biological and pathological processes likely through its regulatory roles in collagen fibrillogenesis, intermolecular cross-linking, and collagen-cell interaction. Recently, with the advances in molecular/cell biology and analytical chemistry, the molecular mechanisms of collagen glycosylation have been gradually deciphered, and the type and extent of glycosylation at the specific molecular loci can now be quantitatively analyzed. In this chapter, we describe quantitative analysis of collagen glycosylation by high-performance liquid chromatography (HPLC) and semiquantitative, site-specific analysis by HPLC-tandem mass spectrometry.

Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.

Fibrillar type I collagen is the major organic component in bone, providing a stable template for mineralization. During collagen biosynthesis, specific hydroxylysine residues become glycosylated in the form of galactosyl- and glucosylgalactosylhydroxylysine. Furthermore, key glycosylated hydroxylysine residues, alpha 1/2-87, are involved in covalent intermolecular cross-linking. Although cross-linking is crucial for the stability and mineralization of collagen, the biological function of glycosylation in cross-linking is not well understood. In this study, we quantitatively characterized glycosylation of non-cross-linked and cross-linked peptides by biochemical and nanoscale liquid chromatography-high resolution tandem mass spectrometric analyses. The results showed that glycosylation of non-crosslinked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving alpha 1/2-87, divalent cross-links were glycosylated with both mono- and disaccharides, whereas the mature, trivalent cross-links were primarily monoglycosylated. Markedly diminished diglycosylation in trivalent cross-links at this locus was also confirmed in type II collagen. The data, together with our recent report (Sricholpech, M., Perdivara, I., Yokoyama, M., Nagaoka, H., Terajima, M., Tomer, K. B., and Yamauchi, M. (2012) Lysyl hydroxylase 3-mediated glucosylation in type I collagen: molecular loci and biological significance. J. Biol. Chem. 287, 22998-23009), indicate that the extent and pattern of glycosylation may regulate cross-link maturation in fibrillar collagen.

Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib-/- mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2-11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib-/- fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.

The most abundant proteins in vertebrates - the collagen family proteins - play structural and biological roles in the body. The predominant member, type I collagen, provides tissues and organs with structure and connectivity. This protein has several unique post-translational modifications that take place intra- and extra-cellularly. With growing evidence of the relevance of such post-translational modifications in health and disease, the biological significance of O-linked collagen glycosylation has recently drawn increased attention. However, several aspects of this unique modification - the requirement for prior lysyl hydroxylation as a substrate, involvement of at least two distinct glycosyl transferases, its involvement in intermolecular crosslinking - have made its molecular mapping and quantitative characterization challenging. Such characterization is obviously crucial for understanding its biological significance. Recent progress in mass spectrometry has provided an unprecedented opportunity for this type of analysis. This review summarizes recent advances in the area of O-glycosylation of fibrillar collagens and their characterization using state-of-the-art liquid chromatography-mass spectrometry-based methodologies, and perspectives on future research. The analytical characterization of collagen crosslinking and advanced glycation end-products are not addressed here.

The most abundant proteins in vertebrates – the collagen family proteins – play structural and biological roles in the body. The predominant member, type I collagen, provides tissues and organs with structure and connectivity. This protein has several unique post-translational modifications that take place intra- and extra-cellularly. With growing evidence of the relevance of such post-translational modifications in health and disease, the biological significance of O-linked collagen glycosylation has recently drawn increased attention. However, several aspects of this unique modification – the requirement for prior lysyl hydroxylation as a substrate, involvement of at least two distinct glycosyl transferases, its involvement in intermolecular crosslinking – have made its molecular mapping and quantitative characterization challenging. Such characterization is obviously crucial for understanding its biological significance. Recent progress in mass spectrometry has provided an unprecedented opportunity for this type of analysis. This review summarizes recent advances in the area of O-glycosylation of fibrillar collagens and their characterization using state-of-the-art liquid chromatography–mass spectrometry-based methodologies, and perspectives on future research. The analytical characterization of collagen crosslinking and advanced glycation end-products are not addressed here.

Introduction: Recessive osteogenesis imperfecta (OI) is caused by mutations in genes encoding proteins involved in post-translational interactions with type I collagen. Types VII–IX OI involve defects in the collagen prolyl 3-hydroxylation complex, which modifies α1(I)Pro986. PPIB encodes CyPB, a complex component with PPIase activity and the major isomerase facilitating collagen folding. We investigated the role of CyPB in collagen post-translational modifications and crosslinking. Methods: Ppib KO mice were generated using a gene-trap ES cell clone with a β-geo reporter inserted into Ppib intron 1. Type I collagen modifications were analyzed by LC–MS/MS and HPLC. Bone architecture was investigated by micro-CT and DXA. Results: Ppib transcripts and protein are absent in skin, fibroblasts, femora, and calvarial osteoblasts; only residual (<10%) α1(I)P986 3-hydroxylation is detectable in fibroblast and osteoblast collagen. Although collagen from KO cells has delayed electrophoretic mobility, total collagen 5-lysyl and prolyl 4-hydroxylation was normal, suggesting altered glycosylation in KO. MS analyses indicated that, except for lysyl residues involved in crosslinking, most helical residues of KO FB and OB collagen have increased diglycosylation. Total mature crosslinks (HP+LP) in KO bone were increased 1.5–1.7× vs WT. We detected a 4–5-fold increase in trivalent LP crosslinks (P=0.001), which decreased the HP:LP ratio correspondingly. Total immature cross-links (DHLNL+HLNL) were also significantly increased and the DHLNL:HLNL ratio decreased, implying reduced lysyl hydroxylation of helical crosslink residues in KO bone. Abnormal collagen modification is associated with 70–80% reduction of collagen deposited into KO matrix in culture, associated with smaller long bones with significantly reduced BMD, BV and TbN. Conclusions: In Ppib KO mice, absence of CyPB delays collagen folding and alters collagen glycosylation patterns in culture; tissue investigations are ongoing to confirm these effects. Altered modification may impair collagen matrix interactions and promote abnormal bone mineralization. Collagen crosslink patterns are shifted to trivalent forms lacking helical Hyl, possibly contributing to decreased matrix deposition and bone strength.

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids –(X—Y—Gly)n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides (i.e., in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed). The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways—amide bond and glycosidic bond cleavage—are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e., Arg, Lys, HyK, and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides. Figure ᅟ

Introduction: Collagen glycosylation occurs at specific hydroxylysine (Hyl) residues producing galactosyl-Hyl (G-Hyl) and glucosylgalactosyl-Hyl (GG-Hyl). The glycosylation pattern is tissue specific and its alteration has been implicated in bone disorders. However, the molecular mechanism of this modification is still not well understood. Recently, we identified a novel glycosyltransferase 25 domain 1 (Glt25d1) expressed in mouse osteoblastic cell line, MC3T3-E1 (MC). Objective: To elucidate the role of Glt25d1 in bone type I collagen modification by employing a loss-of-function approach in vitro. Methods: MC-derived clones stably suppressing Glt25d1 (Sh clones) were generated by short-hairpin RNA technology. Type I collagen was purified from the Sh clones and from two controls, MC and those transfected with an empty vector. The site-specific glycosylation pattern of Sh type I collagen was characterized and compared with that of controls by mass spectrometry (MS). Aliquots of matrices were reduced with NaB3H4 and subjected to quantitative cross-link analysis. Collagen fibrils in the cultures were also analyzed by transmission electron microscopy. Results: Single cell-derived clones stably suppressing Glt25d1 (by ~90-95%) were successfully generated. Five glycosylation sites were identified by MS with the predominant site found as the cross-linking site. In Sh collagen, Hyl glycosylation (GG-Hyl + G-Hyl) was found significantly decreased, with a concomitant increase in non-glycosylated Hyl. Cross-link analysis showed that, in Sh clones, dihydroxylisinonorleucine (DHLNL) was slightly decreased with a significant increase in its maturational product, pyridinoline (Pyr). Mean collagen fibril diameters in Sh clones were significantly smaller than controls, and the diameters tended to inversely correlate to the Pyr/DHLNL ratio. Conclusion: Glt25d1 catalyzes galactosylation of Hyl in bone type I collagen. Hyl glycosylation is a negative regulator of collagen cross-link maturation, which may in turn affect collagen fibril growth.

Recently, by employing the short hairpin RNA technology, we have generated MC3T3-E1 (MC)-derived clones stably suppressing lysyl hydroxylase 3 (LH3) (short hairpin (Sh) clones) and demonstrated the LH3 function as glucosyltransferase in type I collagen (Sricholpech, M., Perdivara, I., Nagaoka, H., Yokoyama, M., Tomer, K. B., and Yamauchi, M. (2011) Lysyl hydroxylase 3 glucosylates galactosylhydroxylysine residues in type I collagen in osteoblast culture. J. Biol. Chem. 286, 8846–8856). To further elucidate the biological significance of this modification, we characterized and compared type I collagen phenotypes produced by Sh clones and two control groups, MC and those transfected with empty vector. Mass spectrometric analysis identified five glycosylation sites in type I collagen (i.e. α1,2-87, α1,2-174, and α2-219. Of these, the predominant glycosylation site was α1-87, one of the major helical cross-linking sites. In Sh collagen, the abundance of glucosylgalactosylhydroxylysine was significantly decreased at all of the five sites with a concomitant increase in galactosylhydroxylysine at four of these sites. The collagen cross-links were significantly diminished in Sh clones, and, for the major cross-link, dihydroxylysinonorleucine (DHLNL), glucosylgalactosyl-DHLNL was diminished with a concomitant increase in galactosyl-DHLNL. When subjected to in vitro incubation, in Sh clones, the rate of decrease in DHLNL was lower, whereas the rate of increase in its maturational cross-link, pyridinoline, was comparable with controls. Furthermore, in Sh clones, the mean diameters of collagen fibrils were significantly larger, and the onset of mineralized nodule formation was delayed when compared with those of controls. These results indicate that the LH3-mediated glucosylation occurs at the specific molecular loci in the type I collagen molecule and plays critical roles in controlling collagen cross-linking, fibrillogenesis, and mineralization. Background: Type I collagen is the most abundant organic component in bone, providing form and stability. Results: Lysyl hydroxylase 3-mediated glucosylation occurs at specific sites in collagen, including cross-linking sites, and suppression of this modification results in defective collagen and mineralization. Conclusion: The data indicate the critical importance of this modification in bone physiology. Significance: Alterations of this collagen modification may cause bone defects.