Molecular Characterization of Collagen Hydroxylysine O-Glycosylation by Mass Spectrometry: Current Status

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

Glycosylation and Cross-linking in Bone Type I Collagen

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 glucosylgalactosyl-hydroxylysine. Furthermore, key glycosylated hydroxylysine residues, α1/2-87, 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-cross-linked hydroxylysine is different from that involved in cross-linking. Among the cross-linked species involving α1/2-87, 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

Lysyl Hydroxylase 3-mediated Glucosylation in Type I Collagen: MOLECULAR LOCI AND BIOLOGICAL SIGNIFICANCE

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

Unusual Fragmentation Pathways in Collagen Glycopeptides

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

Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta

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

Strukturaufklärung der mit Autoimmunerkrankungen assoziierten Proteine mittels Hochdruckflüssigkeitschromatographie - Massenspektrometrie

Autoimmunerkrankungen haben in den letzten Jahren zunehmende medizinische Bedeutung aufgrund der zunehmeden Zahl individueller Erkrankungen und der geringen Kenntnisse über Krankheitsursache(n) erlangt. Eindeutige Hinweise auf die Bedeutung des Immunsystems für Pathogenese und Verlauf von Autoimmunerkrankungen wurden erhalten, die derzeit zur Entwicklung von neuen therapeutischen Ansätzen führen; künftige Fortschritte in der Entwicklung und Herstellung von wirksamen Vaccinen werden jedoch wesentlich von dem besseren Verständnis der strukturellen Grundlagen des Immunsystems abhängen. Die Massenspektrometrie hat sich in den letzten Jahren aufgrund ihrer hohen Nachweisempfindlichkeit, Genauigkeit der Massenbestimmung, schnellen Analysenzeiten und Anwendbarkeit zur Analyse komplexer Gemische als Hochleistungsmethode für Strukturaufklärung von Proteinen und Charakterisierung von Protein-Interaktionen erwiesen.Ein zentrales Merkmal der Alzheimer-Krankheit ist die Bildung und Ablagerung von Aggregaten des ß-Amyloid (Aß) im Gehirn. Die beiden ersten Abschnitte der vorliegenden Dissertation behandeln die Aufklärung der vollständigen Primärstukturen sowie Identifizierung der N-Glykosilierung von Aß-spezifischen Antikörpern. Plaque-spezifische Antikörper erkennen ein N-terminales Aß-Epitop. Mittels HPLC-Tandem-Massenspektrometrie (LC-MS/MS) konnte die vollständige Struktur eines Plaque-spezifischen monoklonalen Maus-Antikörpers aufgeklärt werden. Nahezu vollständige Sequenzbestimungen wurden mittels Datenbank-Suchverfahren sowie De novo - Sequenzierung für die schweren und leichten Ketten erhalten, insbesondere die vollständige Aufklärung von 5 der 6 CDR-Sequenzen des Antikörpers. Die Glycosilierung im Fc-Teil des Antikörpers wurde als Komplexstruktur-Typ mit bis zu 4 terminalen Galactosylresten und einem geringen Anteil an N-Glycolyl-Neuraminsäure identifiziert.Physiologische humane Aß-Antikörper ( Plaque-protektive Antikörper) wurden im Plasma von gesunden Personen identifiziert, die ein Epitop im C-terminalen Bereich (Aß-(21-37)) erkennen und die Bildung von Aß-Aggregaten inhibieren. Die vollständige Primärstruktur und Glykosilierung eines Plaque-protektiven monoklonalen Antikörpers der Maus gegen ein Aß(17-24) Epitop wurde mittels LC-MS/MS, in Kombination mit Stoss-induzierter Dissoziation (CID) und Elektronentransfer- Dissoziation (ETD) aufgeklärt. Die Strukturen aller 6 CDR-Domänen sowie eine bisher unbekannte Konsensus-Sequenz der Glycosilierung in den leichten Ketten konnten bestimmt werden. Die Glycosilierungsstruktur enthält teilweise hybridisierte Glycane der Mehrantennenstruktur mit einem High-mannose -Typ und einem Komplex-Typ, und terminalen N-Acetyl- und N-Glycolyl-Neuraminyl-Gruppen.Weitere Strukturuntersuchungen wurden von humanen, Plaque-protektiven Aß-Autoantikörpern aus Immunglobulinpräparaten nach Affinitätsisolierung mit immobilisiertem Aß(12-40) durchgeführt. Durch Bestimmung der Primärstrukturen konnten alle 4 IgG-Subklassen auf der Ebene der entsprechenden Glycopeptide bestimmt werden. Die Subklassen-spezifische Glykosilierung ergab relativ erhöhte Anteile an IgG2/IgG3 sowie IgG4. Die Bestimmung der Glykanstruktur der einzelnen IgG-Subklassen ergab ähnliche Glycosilierungen mit leicht erhöhtem Agalactosylanteil.ß-Amyloid wird durch proteolytische Prozessierung des Transmembran-Amyloid-Vorläuferproteins APP gebildet, dessen genaue Struktur und Funktionen bisher nicht bekannt ist. In Strukturuntersuchungen im Rahmen der vorliegenden Arbeit konnten 3 spezifische O-Glycosilierungen an den Threoninresten T291, T292 und T576 mittels LC-MS/MS in Kombination mit ETD- und CID- Fragmentierung aufgeklärt werden. Die O-Glycosilierungen wurden als kurze Core-1 Typ- Glycane mit N-Acetylgalactosamin und terminalen Silinsäureresten, sowie linearen (T291, T292) und verzweigten Strukturen (T576) identifiziert.Im letzten Abschnitt der Arbeit wurde die Subklassen-spezifische Glycosilierung von Plasma-Immunglobulin bei Patienten mit Myositose, im Rahmen einer klinisch-diagnostischen Studie zur Ermittlung von Risikofaktoren bei Patienten mit systemisch-rheumatischen Erkrankungen untersucht. Plasma IgG wurde von Patienten, gesunden Zwillingen sowie Kontrollgruppen gleichen Alters mittels Protein G-Affinitätschromatographie isoliert. Die Analyse der Glycosilierungsstruktur ergab statistisch signikante Erhöhungen von fucosylierten Agalactosyl-Glycoformen bei Patienten im Vergleich zur Kontrollgruppe. Diese Ergebnisse deuten auf eine genetische Prädisposition für die Entwicklung von Autoimmun-Erkrankungen, möglicherweise aufgrund von Umweleinflüssen

Site Specific Identification of N-Linked Glycosylation in Proteins by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

Recently, we reported the characterization of the glycans attached at the 11 N-glycosylation sites of Hepatitis C virus E2 envelope glycoprotein by tandem mass spectrometry. Infections caused by Hepatitis C virus represent the main cause of liver diseases such as hepatitis, cirrhosis and hepatocellular carcinoma. The N-linked sugars consist primarily of high mannose glycans, with structures ranging from the minimal core structure, Man3GlcNAc2 (Man3) up to 12 hexose residues attached to the GlcNAc-ß(l 4)-GlcNAc core (depicted as Hex3Man9GlcNAc2). Furthermore, the site N41 (N423) was observed to contain complex type glycans with the structures Man3-GlcNAc and Man3-GlcNAcFuc, in addition to the high mannose population Man3 through Man6, while the site N48 (N430) was occupied exclusively with complex type glycans (Man3-Fuc, Man3-GlcNAcFuc and Man3-GlcNAc2Fuc). The present contribution summarizes our experimental observations upon the factors which may have an impact on the CID tandem mass spectra of glycopeptides

Determination of primary structure and microheterogeneity of a beta-amyloid plaque-specific antibody using high-performance LC tandem mass spectrometry

Using the bottom-up approach and liquid chromatography (LC) in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-β-amyloid (1 17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high-performance LC tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10 antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyroglutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig VL genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core-fucosylated, biantennary, complex-type structures containing zero to two galactose residues

Enhanced electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry of long-chain polysaccharides

A novel strategy was developed to extend the application of electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to the analysis of long-chain polysaccharides. High molecular weight polydisperse maltodextrins (poly-(1-4) glucose) and dextrans (poly-(1-6) glucose) were chosen as model compounds in the present study. Increased ionization efficiency of these mixtures in the positive ion mode was achieved upon modification of their reducing end with nitrogen-containing groups. The derivatization method is based on the formation of a new CN bond between 1,6-hexamethylenediamine (HMD) and the reducing end of the polysaccharide, which exists in solution as an equilibrium between the hemiacetal and the open-ring aldehyde form. To achieve the chemical modification of the reducing end, two synthetic pathways were developed: (i) coupling of HMD by reductive amination and (ii) oxidation of the hemiacetal to lactone, followed by ring opening by HMD to yield the maltodextrin lactonamide of 1,6-hexanediamine (HMMD). Amino-functionalized polysaccharides were analyzed by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR-MS) in the positive ion mode by direct flow injection. The hexamethylenediamine (HMD) and maltodextrin lactonamide of 1,6-hexanediamine (HMMD) moieties provide increased proton affinities which dramatically improve the detection of the long-chain polysaccharides by FTICR-MS. The present approach allowed for identification of single components in mixtures with prominent heterogeneity in the degree of polymerization (DP), without the need for chromatographic separation prior to MS. The high mass accuracy was essential for the unambiguous characterization of the species observed in the analyzed mixtures. Furthermore, molecular components containing up to 42 glucose residues were detected, representing the largest polysaccharide chains analyzed so far by ESI FTICR-MS