Glycosylation and Cross-linking in Bone Type I Collagen*

Background: Bone type I collagen is glycosylated. Results: The major glycosylation sites are involved in intermolecular cross-linking. The extent and pattern of glycosylation vary depending on the site, type, and maturation of cross-links. Conclusion: Glycosylation may control collagen cross-linking in bone type I collagen. Significance: The results provide important insight into the role of glycosylation in collagen stability in bone. 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.

Despite current knowledge regarding type I collagen glycosylation (14,30), the precise molecular loci and the extent/type of glycosylation in cross-linked and non-cross-linked residues are still not well characterized. This information, however, is essential to understand the role of glycosylation in collagen biosynthesis and function. Mass spectrometry (MS) has become increasingly used in the structural characterization of collagens from different sources, and several studies have utilized MS to characterize collagen cross-linked peptides (30 -34). Although these studies pioneered the MS-based characterization of collagen cross-linked species, their major limitation is the use of low resolution mass analyzers. This renders accurate characterization of species bearing naturally occurring heterogeneity (e.g. incomplete hydroxylation of Lys or Pro) or of species with similar mass/charge ratio (m/z), difficult. In the present study, we used high performance/high resolution nanoscale liquid chromatography-tandem mass spectrometry (nanoLC/MS/ MS) to comprehensively characterize the glycosylation at various molecular loci in non-cross-linked and cross-linked peptides in bovine bone type I collagen. A multistep chromatographic approach was employed to obtain highly purified crosslinked tryptic peptides. The analytical challenges associated with these large cross-linked species were overcome with the use of an alternative enzyme. The molecular distribution of glycosylation in non-cross-linked Hyl and immature and mature cross-links was quantitatively determined by nanoLC/ MS. The results revealed a differential glycosylation pattern, depending on the involvement in cross-linking, molecular loci, and type and maturational stage of cross-linking.

EXPERIMENTAL PROCEDURES
Collagen Preparation-Fresh femoral bone samples from 2-3-year-old bovine animals were obtained commercially (Aries Scientific, Dallas, TX). After removing the surrounding connective tissues, both ends of the bone, and the bone marrow, the bones were cut into small pieces. All operations were carried out at 4°C. The bone pieces were pulverized to a fine powder under liquid nitrogen using a Spex Freezer Mill (Spex, Inc., Metuchen, NJ). Pulverized samples were washed several times with cold phosphate-buffered saline (PBS), and cold distilled water, centrifuged at 4000 ϫ g for 30 min, and lyophilized. Bone powder was then demineralized with 0.5 M EDTA, pH 7.5, for 2 weeks with several changes of the EDTA solution by centrifugation at 4000 ϫ g. The EDTA-insoluble residue was thoroughly washed with cold distilled water by repeated centrifugation at 4000 ϫ g and lyophilized.
Reduction with NaB 3 H 4 -Demineralized bone (ϳ2.0 g) was suspended in buffer containing 0.15 M N-trismethyl-2-amino-ethanesulfonic acid and 0.05 M Tris-HCl, pH 7.4, and reduced with standardized NaB 3 H 4 . The specific activity of the NaB 3 H 4 was determined by the method described previously (35,36). The reduced samples were washed with cold distilled water several times by repeated centrifugation at 4000 ϫ g and lyophilized. Upon reduction, the dehydrodihydroxylysinonorleucine (dehydro-DHLNL) and dehydrohydroxylysinonorleucine (dehydro-HLNL) and their respective keto amine forms are reduced to stable secondary amines, DHLNL and HLNL, and radiolabeled simultaneously (ϩ2 Da molecular mass increase). Hereafter, the terms DHLNL and HLNL will be used for both the unreduced and reduced forms.
Cross-link Analysis-Reduced collagen was hydrolyzed with 6 N HCl and subjected to cross-link analysis as described previously (37). The reducible cross-links were analyzed as their reduced forms (i.e. DHLNL and HLNL, respectively). The levels of the immature reducible (DHLNL and HLNL) and mature non-reducible cross-links (Pyr and d-Pyr) were quantified and expressed in mol/mol of collagen.
Digestion with Trypsin-Digestion with trypsin of reduced bone collagen was prepared by the procedure described previously (37) with slight modifications. Briefly, the reduced collagen was heated at 65°C for 15 min, digested with 1% (w/w) trypsin for 16 h at 37°C, reheated to 65°C for 10 min, and retreated with 0.5% (w/w) trypsin for 3 h at 37°C. Over 99% of the starting material was recovered in the supernatant of the trypsin digest.
Molecular Sieve Chromatography-Molecular sieve chromatography of the bone tryptic digest was performed on a HiLoad Superdex 75 preparative scale column (1.6 ϫ 60 cm) equilibrated with 0.05 M ammonium bicarbonate (pH 7.9) at room temperature. Aliquots of the reduced bone tryptic digest (ϳ200 mg) were injected and separated at a flow rate of 0.75 ml/min. Fractions of 1.5 ml were collected, and their absorbance (230 nm), radioactivity, and fluorescence (excitation 330 nm and emission 390 nm) were measured. Fluorescence measurement was employed to detect Pyr and d-Pyr, and radioactivity was used to detect DHLNL and HLNL. The main fluorescent (F1-F3) and radioactive (R1 and R2) fractions were recovered. An equal aliquot from each fraction was lyophilized and subjected to cross-link analysis as described above. The majority of aliquots were subjected to further purification by reversed phase chromatography or digestion with chymotrypsin (see below). To identify fractions containing the Prl cross-link, aliquots from each fraction were subjected to Ehrlich's chromogen (EC) analysis following reported methods (30,38). Briefly, the aliquots were lyophilized and resuspended in distilled water, and 35 l of a 5% solution (w/v) of p-dimethylaminobenzaldehyde in 4 M perchloric acid were added. The reaction was incubated for 5 min at room temperature. The absorbance (572 nm) was monitored with a F-2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The main EC-positive fractions (P1-P3) eluted at similar positions as F1-F3 and were further characterized by mass spectrometry (see below).
Chymotrypsin Digestion-Chymotrypsin digestion of the fractions F1-F3, R1, and R2 collected after molecular sieve chromatography was performed as follows. The lyophilized fractions were dissolved in 100 l of 25 mM ammonium bicarbonate (pH 7.4). Aliquots of 50 l from each fraction were incu-bated with 2 l of chymotrypsin solution (0.5 g/l in 25 mM ammonium bicarbonate) overnight at room temperature. These digests were further analyzed by nanoLC/MS/MS.
Reversed Phase High Performance Liquid Chromatography-The lyophilized fluorescent and radioactive fractions resolved by molecular sieve chromatography were further fractionated by reversed phase on a SOURCE 5RPC ST 4.6/150 column (GE Healthcare) using a Varian HPLC system (Prostar 240/310, Varian, Walnut Creek, CA). The solvents employed were 2% (v/v) acetonitrile in 10 mM ammonium acetate (solvent A) and 70% acetonitrile in deionized water (v/v) (solvent B). The samples were dissolved in solvent A and eluted with a linear gradient from 0 to 15% solvent B for the first 10 min, followed by a linear gradient from 15 to 50% solvent B over the next 50 min at a flow rate of 1.0 ml/min at room temperature. The effluent was monitored for absorbance (230 nm) and fluorescence (excitation 330 nm, emission 390 nm). Fractions of 1 ml were collected, and aliquots were subjected to radioactivity and fluorescence measurements. The radioactive and fluorescent fractions were pooled, lyophilized, and subjected to further analysis.
Ion-Exchange Chromatography-Ion-exchange chromatography of fractions collected after reversed phase separation was performed on a TSK-GEL DEAE-5PW column (8.0 ϫ 75 mm, Tosoh Bioscience LLC, Montgomeryville, PA). The column was equilibrated with 0.01 M NH 4 HCO 3 containing 1% isopropyl alcohol. Elution was carried out with a linear gradient from 0.01 to 0.25 M NH 4 HCO 3 at a flow rate of 1 ml/min, monitoring the absorbance at 230 nm. One-minute fractions were collected, and those corresponding to fluorescent and/or radioactive signals were pooled and lyophilized. Their structural characterization was performed by mass spectrometry.
Nanoscale Liquid Chromatography, Mass Spectrometry, and Data Analysis-Glycosylated, non-cross-linked collagen peptides were characterized by nanoLC/MS/MS from a bovine bone tryptic digest on a nanoACQUITY UPLC-Q-Tof Premier mass spectrometer (Waters, Milford, MA). Their structureand site-specific quantitative analyses were performed as described previously (24).
Flow Injection Analyses of Purified Cross-linked Tryptic Peptides-Flow injection analyses of purified cross-linked tryptic peptides were performed by positive ion nanoelectrospray (ESI ϩ ) on a Waters Micromass Q-Tof Micro mass spectrometer (Waters). The samples were desalted using a C18 ZipTip pipette tip (EMD Millipore, Billerica, MA) and reconstituted in 50% acetonitrile with 0.1% formic acid (v/v). Dilutions were performed as needed to ensure optimal signal intensity. Mass spectrometer parameters were as follows: capillary voltage, 3.8 kV; sampling cone, 30; source temperature, 80°C; desolvation temperature, 20°C.
NanoLC/MS/MS Analyses-NanoLC/MS/MS analyses of cross-linked peptides were carried out on a nanoACQUITY UPLC-Q-Tof Global mass spectrometer (Waters) with datadependent acquisition and charge state selection of the top four ions. Separations of chymotryptic digests of fractions after molecular sieve chromatography were carried out on a C18 BEH column (1.7 m, 75 m ϫ 100 mm) at 0.3 l/min, with a gradient from 1 to 50% solvent B over 30 min. The solvents were as follows: solvent A (0.1% formic acid in deionized water) and solvent B (0.1% formic acid in acetonitrile). Mass spectrometer settings for (ϩ)-nano-ESI were as follows: capillary voltage, 3.4 kV; sampling cone, 30; source temperature, 80°C. To ensure optimal fragmentation, the collision energies were optimized over the range 20 -30 V.
Data Analysis-Data analysis was performed using the Mass-Lynx software, version 4.1 (Waters), including the embedded deconvolution algorithms MaxEnt 1 and 3. The glycoform distribution of C-telo-derived cross-linked peptides was determined from the LC/MS data of chymotryptic digests. Quantitative glycosylation analyses (percentage of free, galactosyl (G), and glucosylgalactosyl (GG)) of cross-linked peptides were performed as described for non-cross-linked peptides by integrating the extracted ion chromatograms (EICs) generated post-data acquisition for each charge state of individual glycoforms (24).
Glycosylation of Type II Collagen-derived Cross-links-Articular cartilage from tibial plateau of 0 -1-year bovine knees were obtained using a 5-mm biopsy punch within 24 h of slaughter. The cartilage pieces were rinsed in cold PBS and stored at Ϫ20°C in a protease inhibitor solution until use. The cartilage pieces were then pulverized, washed several times with cold PBS and cold distilled water thoroughly, and lyophilized. The dried samples were reduced with NaB 3 H 4 , digested with trypsin, and fractionated by molecular sieve chromatography as described above. The cross-link-containing fractions were  identified by cross-link analysis and then subjected to nanoLC/ MS/MS as described above.
Two additional helical Hyl residues involved in cross-linking are ␣1-930 and ␣2-933. The tryptic peptide containing ␣1-930 spans the residues 928 GI-Hyl 930 -GHR 933 . This peptide was not Isolation, Molecular Characterization, and Glycosylation of Cross-linked Peptides-Using a NaB 3 H 4 -reduced collagen tryptic digest as starting material, a multistep chromatographic approach was employed to obtain highly purified tryptic crosslinked peptides. Three sequential chromatographic steps were performed: 1) molecular sieve; 2) reversed phase; and 3) ion exchange. After each step, the fractions containing immature and mature cross-linked peptide were detected based on radioactivity and fluorescence, respectively (Fig. 2), whereas Prl cross-links were detected by reaction with EC (see "Experimental Procedures") (40). The chromatographic profiles are shown in Fig. 2, with the major fluorescent and radioactive peaks designated as F1-F3 and R1 and R2, respectively. Cross-link analysis performed after molecular sieve separation was in agreement with the profiles shown in Fig. 2 (i.e. Pyr/d-Pyr were found in fractions F1-F3, whereas DHLNL/HLNL were found in R1 and R2). Quantitative cross-link analysis on bone collagen (Table 2) indicated that DHLNLs are the most abundant (1.58 mol/mol of collagen), followed by HLNL (0.50 mol/mol) and Pyr (0.28 mol/mol), whereas d-Pyr was found in low amounts (0.04). Based on these numbers, the molecular loci identified in F1-F3 and R1 and R2 (i.e. R1 and F1 contain the C-telo derived cross-links; see below), and direct cross-link analyses of these     fractions, we estimated that within DHLNLs, the C-telo-derived (␣1/2-87-involved) cross-link was ϳ4-fold higher than that of the N-telo-derived (ϳ1.3 versus ϳ0.3), whereas the levels of C-and N-telo-derived HLNLs were comparable. Pyr was almost equally located between C-and N-telo, whereas d-Pyr was enriched in the N-telo (ϳ80% of total d-Pyr). The relative small fluorescence of the N-telo Pyr/d-Pyr (F2 and F3) in Fig. 2 is probably due to the fluorescence quenching (30). From F1 and R1 fractions, after the final chromatographic step, one fluorescent (a) and two radioactive peaks (b and c) were collected (Fig. 2, D-F) and were analyzed by flow injection nano-ESI-MS.

Flow Injection Analysis-Mass Spectrometry of Peaks a, b, and c-
The theoretical molecular weights of collagen cross-linked peptides were determined using the equations listed in Table 3. The raw mass spectrum of the cross-linked species contained in peak a is shown in Fig. 3A. The charge state envelope containing the ions of m/z 2532.62 (4ϩ), 2026.27 (5ϩ), 1688.75 (6ϩ), 1447.65 (7ϩ), 1266.81 (8ϩ), and 1126.14 (9ϩ) was deconvoluted to the average mass of 10,126.4 Da (Fig. 3A, inset). This value is consistent with the theoretical average molecular weight of G-Pyr cross-linked tryptic peptides ␣1-(993-22 C ) ϫ ␣1-(993-22 C ) ϫ ␣1-(76 -90). The peptide identities were confirmed by MS/MS (not shown). Ion clusters of species with a molecular mass of 162 Da lower or higher than G-Pyr, corresponding to Pyr and GG-Pyr, respectively, were observed with very low relative abundance, indicating that G is the predominant glycoform of the C-telo-derived Pyr cross-link.
The deconvoluted mass spectra of the DHLNL cross-linked peptides ␣1-(993-22 C ) ϫ ␣1-(76 -90) and ␣1-(993-22 C ) ϫ ␣2-(76 -90) observed in peaks b and c are shown in Fig. 3, B and C, and Supplemental Fig. S2, respectively. The ratio of the former to the latter was ϳ3:1 (Fig. 2C), which is consistent with our previous report (36). In contrast to Pyr, both the G and GG glycoforms of ␣1-/␣2-87-containing DHLNLs represent major species. As shown in Fig. 3, B and C, extended molecular het- erogeneity of the cross-linked peptides was observed as Ϯ16 Da species. The ϩ16 Da might be due to oxidized Met-86 in the ␣1 h peptide, whereas the Ϫ16 Da could be attributed to either incomplete Pro hydroxylation or HLNL species, respectively. The occurrence of the latter was confirmed in the fractions b and c (i.e. when the fractions were hydrolyzed and directly ana-lyzed for cross-links, HLNL was present as a minor species in both fractions). These low abundance species are difficult to assign because their MS/MS lack informative fragment ions. Nevertheless, the species of molecular mass 5791.96 Da (Fig.  3C) probably represent the HLNL (␣1-16 C ϫ ␣2-87). This would be in agreement with a previous study suggesting higher FIGURE 5. A-G, EICs showing the relative glycoform distribution (free, G, and GG) for various C-telopeptide-containing immature and mature cross-linked peptides observed in the LC/MS-MS analyses of chymotryptic digests of fractions F1 and R1 collected after molecular sieve separation. For each cross-linked species, the most abundant charge state was used to generate the EICs. For a particular cross-linked species, the EICs were normalized to that of the most abundant glycoform.
Quantitative Determination of Glycosylation in the C-telopeptide Containing Immature and Mature Cross-linked Peptides-For LC/MS-based quantitative glycosylation analysis of the C-telo cross-linked peptides, the multistage chromatographic approach was modified as follows. 1) the NaB 3 H 4 -reduced bone tryptic digest was separated by molecular sieve chromatography as described above. Fractions were collected based on fluorescence (F1-F3) and radioactivity (R1 and R2). 2) the tryptic peptide fractions F1-F3, R1, and R2 were digested with chymotrypsin to reduce the size of the cross-linked peptides. 3) the resulting double digests were analyzed by nanoLC/MS/MS.
The C-telo DHLNLs were well characterized in both tryptic and chymotryptic forms. The MS/MS spectra of the G-DHLNL ␣1 C ϫ ␣1 h /␣2 h protonated molecules (Fig. 4, C and D, and supplemental Fig. S3, C and D) confirm their structural assignment. In MS/MS, no neutral loss of G was observed, suggesting increased stability of the glycosidic bond of cross-linked peptides. Lower abundance glycosylated ions G-and GG-HLNLs were assigned based on the 16 Da lower mass compared with the corresponding DHLNL glycoforms.
To determine the distribution of the free, G, and GG forms in immature/mature C-telo-containing cross-linked species, glycoform EICs were generated from the LC/MS of F1-and R1-chymotryptic digests and were normalized to the most abundant species within a cross-link type (Fig. 5). Quantitative analyses of technical triplicates are shown in Table 4. Within mature cross-links Pyr/Prl ␣1 C ϫ ␣1 C ϫ ␣1 h /␣2 h , the G glycoform is the most abundant, whereas GG is minimal. The rela-tive amounts of ␣1 C free Pyr are higher in ␣2 h compared with ␣1 h . In contrast to mature cross-links, both the G and GG glycoforms of the DHLNL cross-links ␣1 C ϫ ␣1 h /␣2 h are highly abundant, with minute amounts of free DHLNLs. The glycosylation patterns of HLNL ␣1 C ϫ ␣1 h /␣2 h are comparable with those of DHLNLs. However, quantitative analyses of HLNLs and of ␣2 h -Prl glycoforms were not performed because of poor signal/noise ratio.
The relative abundance of the C-telo-/helical residue 87-based Pyr (i.e. the ␣1 h /␣2 h ratio) was estimated from the LC/MS data of chymotryptic F1. The EICs of ␣1 h and ␣2 h G-Pyr (Fig. 6A) suggest that ␣1 h -Pyr is far more abundant (ϳ85%) than ␣2 h -Pyr (ϳ15%). This might explain why the ␣2 h Pyr was not observed previously by flow injection analysis of peaks a, b, and c. Moreover, we determined the relative abundance of Pyr/ Prl containing ␣1-87 to be ϳ4:1 (Fig. 6B and Table 4B).
Characterization of N-telopeptide Containing Immature and Mature Cross-links-By flow injection analysis, the molecular mass of free (non-glycosylated) DHLNL ␣1 N ϫ ␣2 h , determined as 5943.18 Da, is consistent with the structure of the reduced cross-link ␣1 N -(15 N -9) ϫ ␣2 h -(928 -963) containing pyro-Gln in ␣1 N peptide and three Hyp residues and deamidated Asn-936 in the ␣2 h peptide. This assignment was confirmed from the MS/MS of the ion of m/z 991.530 (6ϩ) (Fig. 7A and supplemental Fig. S4, A and B). The ␣1 N ϫ ␣2 h was observed completely non-glycosylated, consistent with the profile of the non-cross-linked peptide ␣2-(928 -963) (see above).

DISCUSSION
Collagen glycosylation, consisting of the O-glycosides in the form of G-and GG-Hyl, is a key modification involved in collagen cross-linking, fibrillogenesis, mineralization, and collagen-protein interactions (1,21,24,29,43,44). Type I collagen, the main organic component in bone, is one of the minimally glycosylated members in the collagen family (45). Even among type I collagen in various tissues, such as skin type I collagen, the extent of glycosylation in bone type I collagen is low (45,46). Despite numerous studies indicating the functional importance of this modification in type I collagen biosynthesis, the type and distribution of Hyl glycosides and their involvement in crosslinking have not been characterized in a comprehensive manner. In this study, by employing a wide range of analytical methods, we performed residue-specific quantitative glycosylation analysis in both non-cross-linked and cross-linked peptide species from bovine bone type I collagen.
The C-and N-telo immature and mature cross-links were characterized in the form of tryptic/chymotryptic cross-linked peptides. In agreement with previous studies of bone collagen (24,36), cross-link analysis suggested that the ϳ80% of DHLNL was derived from the C-telo site. This might represent a feature conserved among species, because a similar distribution was found in mouse osteoblasts as well (24). Cross-linked peptides containing DHLNL ␣1-16 C ϫ ␣1/␣2-87 and HLNL ␣1-16 C ϫ ␣1-87 were found mostly glycosylated in the form of both G and GG glycoforms having similar abundances. In contrast, the N-telo DHLNL ␣1-9 N ϫ ␣1-930, ␣1-9 N ϫ ␣2-933, and ␣2-5 N ϫ ␣2-933 were found non-glycosylated (30). For each cross-linked species, the most abundant charge state (4ϩ for Pyr and 6ϩ for DHLNL, respectively) was used to generate the EICs. Within each set of glycosylated cross-links, the EICs were normalized to that of the most abundant glycoform.
In contrast to the immature cross-links, the mature Pyr/Prl species (␣1-16 C ϫ ␣1-16 C ϫ ␣1/2-87) were found glycosylated primarily as G-Pyr, with minute amounts of the free and GG glycoforms. In this study, we identified the previously undescribed ␣2-87-containing Pyr and Prl peptide. In comparison with the ␣1-87-involved Pyr and Prl, this is a minute species representing ϳ15% of the former. We previously reported the residue ␣1-87 to be the preferential cross-linking site over ␣2-87 (Ͼ3:1) for the formation of C-telo-derived divalent cross-links in bone. Thus, the low abundance of the mature trivalent cross-links is not surprising. This is probably due to the specific molecular packing in the fibril (35), which could be important for collagen mineralization in an orderly fashion (36).
It is generally accepted that two residues of immature crosslinks, DHLNL/HLNL, mature into one residue of trivalent cross-link, Pyr/d-Pyr, with aging (29,43). However, our in vitro incubation study and the report by Saito et al. (47) showed that the decreased level of immature cross-links is disproportionally higher than the increase of Pyr (24). Most likely, the spontaneous non-enzymatic maturation is controlled by the microenvironment, such as presence of mineral (30,48), the glycosylation state of the immature cross-links (35), and the presence of collagen-binding proteoglycans around the cross-linking sites.
The data in the current study showing abundance of both G-and GG-DHLNL/HLNL but the predominance of G-Pyr and G-Prl forms suggests that GG divalent cross-links may not favor maturation into trivalent cross-links. This was supported by the data on type II collagen. In this heavily hydroxylated and glycosylated collagen type (49), we also found that ϳ50% of the Hyl-87 involving DHLNL is in the form of GG-DHLNL, whereas GG-Pyr at the same locus is minimal. Eyre et al. (32) also reported the lack of glycosylated Pyr at this locus (residue 87) in type II collagen. Possibly, the bulky disaccharide structure sterically hinders or delays the condensation reaction to form mature cross-links. Along with this conjecture, it is interesting to note that, for the N-telo-derived cross-links in which no glycosylated forms were found, immature cross-links were significantly lower, and mature cross-links were higher than the heavily glycosylated C-telo-derived cross-links. Possibly, without glycosylation of the helical Hyl, maturation of the N-teloderived cross-links is accelerated. Clearly, further studies are warranted to determine the fate of glycosylated immature cross-links by employing, for instance, an in vitro maturation study (24) using type I and other types of collagen.
In conclusion, this study provides a detailed molecular characterization of glycosylation and its involvement in intermolecular cross-linking in bovine bone type I collagen. Specific molecular loci and the differential glycosylation pattern between the immature and mature cross-links suggest that glycosylation might regulate the cross-link maturation in fibrillar collagen.