Site-specific Quantitative Analysis of Overglycosylation of Collagen in

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Site-specific Quantitative Analysis of Overglycosylation of Collagen in Osteogenesis Imperfecta Using Hydrazide Chemistry and SILAC Yuki Taga,* Masashi Kusubata, Kiyoko Ogawa-Goto, and Shunji Hattori Nippi Research Institute of Biomatrix, Toride, Ibaraki 302-0017, Japan S Supporting Information *

ABSTRACT: We recently developed a novel method for analysis of collagen O-glycosylations, which include galactosylhydroxylysine (GHL) and glucosyl-galactosyl-hydroxylysine (GGHL), using hydrazide chemistry (Taga, Y., et al. Mol. Cell. Proteomics 2012, 11 (6), M111.010397). Here we investigated an overglycosylation model of collagen produced by cultured skin fibroblasts from osteogenesis imperfecta (OI) patients using this method. Many GHL/GGHL sites were identified in normal and OI type I collagens by LC−MS analysis after the glycopeptide purification procedure. Further, relative quantification was performed on each identified glycopeptide using stable isotope labeling by amino acids in cell culture (SILAC). Significant increases of GGHL were observed at respective glycosylation sites of type I collagen in OI, whereas an OI-specific glycosylation site was not found. These results demonstrated that the overglycosylation of type I collagen proceeds only at specific sites, resulting in accumulation of GGHL, rather than because of an increase of nonspecific glycosylation. Although the roles of collagen O-glycosylations in OI and even in normal conditions are still incompletely understood, the location of GHL/GGHL in the collagen sequence is suggested to be important for their functions. KEYWORDS: osteogenesis imperfecta, collagen, overglycosylation, hydrazide chemistry, SILAC

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INTRODUCTION Osteogenesis imperfecta (OI) is a heritable connective tissue disorder caused mainly by a mutation in the type I collagen gene, characterized by brittleness of the bone. O-Glycosylations unique to collagen, which are attached to hydroxylysine (Hyl) and include galactosyl-hydroxylysine (GHL) and glucosylgalactosyl-hydroxylysine (GGHL), increase in type I collagen derived from patients with OI.1−5 Hyl is well-known to be related to cross-link formation, which is critical for the formation of stable collagen fibrils, but the function of GHL/ GGHL has not been clearly elucidated.6−8 Therefore, the biological significance of the glycosylation and the overglycosylation in OI collagen remains unclear. Specific enzymes add the carbohydrates to the Hyl residue lying in the Y position of repeating collagenous X-Y-Gly triplets until collagen folds into a triple-helical conformation in the endoplasmic reticulum.9,10 Thus, when the abnormal structure caused by a mutation in the collagen gene delays the triple helix formation, the prolonged exposure time to post-translational modifying enzymes leads to overmodifications of OI collagen, especially overglycosylation. This has been confirmed by several experimental models of the delay of the triple helix formation.11−14 There are two possible models of collagen overglycosylation in OI. One is due to an increase of nonspecific glycosylation sites in OI collagen. However, it is also presumable that the glycosylation reaction proceeds only at © 2013 American Chemical Society

specific sites from Hyl to GHL and subsequently GGHL. The amounts of GHL/GGHL in OI collagen have only been measured by amino acid hydrolysis, and detailed analysis on each glycosylation site has not been conducted.1−4 Glycopeptide enrichment using hydrazide chemistry was first reported by Zhang et al. for analysis of N-linked glycoproteins.15 In their method, glycopeptides are oxidized by sodium periodate to generate aldehydes in carbohydrates, which are bound to the hydrazide resin by forming hydrazone bonds. After washing the resin, captured N-glycopeptides are released by PNGaseF and subjected to LC−MS analysis. This method enables high sensitivity identification of the glycosylation sites and is used in various glycoproteomic studies.16−19 In addition, some studies have analyzed glycopeptides with a glycan by using acid to release them from the hydrazide resin instead of PNGase F.20,21 We recently developed a novel method to identify the O-glycosylation sites of collagen using hydrazide chemistry, which we refer to as the “hydrazide method”.22 In our method, galactose oxidase is used to generate an aldehyde group in the galactose of GHL/GGHL with the coordinated addition of catalase and horseradish peroxidase (HRP) to enhance the galactose oxidase activity. In addition, formic acid with heating is used to elute the bound GHL/GGHLReceived: January 25, 2013 Published: April 12, 2013 2225

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ammonium acetate for multiple reaction monitoring (MRM) analysis.

containing peptides by cleaving the hydrazone bonds. Using the hydrazide method, many O-glycosylation sites, including unreported ones, were identified in bovine type I and type II collagens by LC−MS analysis, whereas almost all of them were not identified without the glycopeptide purification procedure. Unfortunately, the hydrazide method is assumed to be unsuitable for accurate quantification because of the insufficient collection efficiency of GHL/GGHL. To this end, in this study, we adopted the use of stable isotope labeling by amino acids in cell culture (SILAC), which has been used for precise proteome quantification23,24 and also for site-specific quantification of post-translational modifications (PTMs).25−27 In the present study, we identified many GHL/GGHL sites in type I collagen from culture media of normal and OI skin fibroblasts by LC−MS/MS analysis with the hydrazide method. The identified glycopeptides were subjected to relative quantification using the hydrazide method and SILAC to investigate the overglycosylation model of type I collagen in OI.

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MRM Analysis of Hyl Derivatives

Analysis was performed on a hybrid triple quadrupole/linear ion trap 3200 QTRAP mass spectrometer (AB Sciex) equipped with an electrospray ionization source. The instrument was coupled to an Agilent 1200 Series HPLC system (Agilent Technologies). The hydrolysates were loaded onto a ZICHILIC column (3.5 μm particle size, L × I.D. 150 mm × 2.1 mm; Merck Millipore) at a flow rate of 200 μL/min and separated by a binary gradient as follows: 90% solvent B (100% acetonitrile) for 5 min, linear gradient of 10−95% solvent A (0.1% acetic acid/5 mM ammonium acetate) for 15 min, 95% solvent A for 5 min, and 90% solvent B for 5 min. Data acquisition and analysis were performed using Analyst 1.5.1 (AB Sciex). Capillary voltage was 3 kV, declustering potential was 25 V, heater gas temperature was 600 °C, curtain gas was 15 psi, nebulizer gas was 80 psi, heater gas was 80 psi, and collision energy was 15 V (Hyl), 33 V (GHL), and 45 V (GGHL). The following MRM transitions were monitored: Hyl (m/z 163.0→128.2), GHL (m/z 325.1→128.1), and GGHL (m/z 487.2→128.1).

EXPERIMENTAL SECTION

Cell Culture

Skin fibroblasts from OI patients [OI-1, G946C substitution in α1 (I), newborn;28 OI-2, probable substitution at an unidentified site in type I collagen, 4 years old] and a normal subject (newborn) were maintained in DMEM (SIGMA) supplemented with 0.5% penicillin-streptomycin (MP Biomedicals) and 10% FBS (Intergen). Cells were incubated at 37 °C in a humidified 5% CO2 incubator.

Purification and Identification of GHL/GGHL Peptides

Purification of GHL/GGHL-containing peptides using the hydrazide method was performed as previously described.22 Briefly, the collagen samples were denatured at 60 °C for 30 min in the reaction buffer (100 mM sodium phosphate, pH 7.2, containing 150 mM NaCl) and digested by tosyl phenylalanyl chloromethyl ketone-treated trypsin (SIGMA). After the reaction, the trypsin was inactivated by heating and addition of trypsin-chymotrypsin inhibitor (SIGMA). Subsequently, the samples were gently mixed with galactose oxidase (30 U; PN: G7907, SIGMA), which was immobilized on CNBr-activated Sepharose 4B (GE Healthcare), catalase (115 U; SIGMA), and HRP (1.5 U; SIGMA) at 37 °C for 24 h. The oxidized samples were coupled to hydrazide resin (Bio-Rad) at pH 4−5 with gentle mixing at 37 °C for 6 h. After extensive washing of the resin with 1.5 M NaCl, 100% methanol, and distilled water, the captured glycopeptides were eluted with 0.1% formic acid by heating at 80 °C for 30 min. The eluted peptides were reduced to their original form using 1 mM sodium borohydride at room temperature for 1 h in alkaline conditions adjusted by triethylamine. The sample solutions were then acidified with formic acid and subjected to LC−MS/MS analysis. Peptide identification was performed on a 3200 QTRAP mass spectrometer coupled to an Agilent 1200 Series HPLC system. The samples were loaded onto an Ascentis Express C18 column (2.7 μm particle size, L × I.D. 150 mm ×2.1 mm; Supelco) at a flow rate of 200 μL/min and separated by a binary gradient as follows: 98% solvent A (0.1% formic acid) for 5 min, linear gradient of 2−50% solvent B (100% acetonitrile) for 15 min, 90% solvent B for 5 min, and 98% solvent A for 5 min. The eluting peptides were analyzed by the information-dependent acquisition method that was operated by selecting the two most intense precursor ions of the prior survey MS scan and then subjecting the precursor ions to MS/ MS fragmentation. The collision energy was automatically determined based on the mass and charge state of the precursor ions using rolling collision energy. Capillary voltage was 5.5 kV, declustering potential was 30−50 V, heater gas temperature was 600 °C, curtain gas was 40 psi, nebulizer gas was 50 psi, and heater gas was 80 psi. The MS and MS/MS scans were

Biochemical Analysis of Collagen

Collagen was purified from the culture medium of the cells incubated in DMEM supplemented with 2% FBS and 200 μM L-ascorbic acid phosphate magnesium salt n-hydrate (Wako) for 3 days.29 The collected culture medium was digested by pepsin (0.1 mg/mL in 0.1 N HCl; SIGMA) at 4 °C for 16 h. The collagen fraction was then precipitated with 1 M NaCl on ice for 3 h. The sample was centrifuged at 16000× g for 10 min at 4 °C, and the precipitated collagen was washed with 1 M NaCl and dissolved in 5 mM acetic acid. The collagen samples were subjected to SDS-PAGE under both reducing and nonreducing conditions using 5% gels. The protein bands were stained with Coomassie Brilliant Blue R250. The relative amounts of the collagens were estimated by the band intensities. Thermal stability of the collagens was measured by a Jasco600 spectropolarimeter (Japan Spectroscopic). Circular dichroism ellipticity at 221 nm was monitored by increasing the temperature from 20 to 50 °C at a constant rate (0.25 °C/ min). The denaturation temperature was defined as the temperature of the midpoint of ellipticity between 20 and 50 °C. Amino Acid Analysis

Equal amounts of the collagen samples were subjected to amino acid hydrolysis. Alkaline hydrolysis was performed with 2 N NaOH at 110 °C for 20 h under N2.30 Subsequently, the samples were neutralized with 30% acetic acid on ice and cleaned on a mixed-mode cation-exchange sorbent (Oasis MCX; Waters). The samples were then dried up. Acid hydrolysis was also performed with 6 N HCl/1% phenol at 110 °C for 20 h in the gas phase under N2. The alkaline and acid hydrolysates were dissolved in 0.1% acetic acid/5 mM 2226

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obtained over the m/z range of 400−1300 and 100−1700, respectively. The acquired MS/MS spectra were searched against the UniProtKB/Swiss-Prot database (release 2011_08, on July 2011) for Homo sapiens species (20244 protein entries) using ProteinPilot software 4.0 (AB Sciex) with the Paragon algorithm.31 Search parameters included digestion by trypsin, biological modifications ID focus, and 95% protein confidence threshold. Default parameters including the number of missed cleavages permitted and mass tolerance for precursor ions and fragment ions were adopted by the software. The probabilities of hydroxylation of Pro and Lys were set higher than those of the defaults, and two residues of galactosyl hydroxylation and glucosyl galactosyl hydroxylation of Lys were added to the search criteria of PTMs. We defined the confidence threshold of the identified peptides to be 90%. SILAC Analysis of GHL/GGHL Peptides

Normal and OI fibroblasts were labeled with light and heavy Lys in SILAC labeling media as described previously.23,24 Briefly, Lys or 13C6-Lys were added to SILAC DMEM media with 2% dialyzed FBS (all from Thermo Scientific). The normal (light) and OI (heavy) cells were incubated in the labeling media with 200 μM ascorbic acid phosphate for 3 days, and the labeled collagens were purified from the culture media. Equal amounts of normal and OI type I collagens, which were estimated by the band intensities from SDS-PAGE analysis, were then combined. GHL/GGHL-containing peptides were purified using the hydrazide method as described above. The glycopeptide solutions were subjected to LC−MS analysis, and relative quantification was conducted by comparing the peak heights of the monoisotopic extracted ion chromatograms (XICs) of the GHL/GGHL peptides between normal and OI collagens. In cases where both peak heights of the light and heavy glycopeptides were difficult to quantify because of their low XIC signals, they were excluded from the quantification analysis.

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Figure 1. Biochemical analysis of normal and OI collagens. Collagens derived from culture media of normal and OI skin fibroblasts were analyzed by SDS-PAGE using 5% gels under (A) reducing and (B) nonreducing conditions. Arrows point to overmodified α1 (I) and α2 (I) of OI collagens. (C) Thermal stability of the collagens was analyzed by circular dichroism. The circular dichroism ellipticity at 221 nm was monitored from 20 to 50 °C. The denaturation temperatures, which were defined as the temperature of the midpoint of ellipticity between 20 and 50 °C, were 41.3 ± 0.5 °C (normal), 39.5 ± 0.4 °C (OI-1), and 39.5 ± 0.1 °C (OI-2). The data represent the mean ± SD of three separate experiments.

RESULTS

Biochemical Analysis of Type I Collagens from Normal and OI Fibroblasts

Table 1. Relative Quantification of Hydroxylysine Derivatives of Type I Collagen from OI Fibroblasts Compared to Normal Fibroblasts by Amino Acid Analysisa

Collagens from culture media of normal and OI (OI-1 and OI2) skin fibroblasts were purified by pepsin digestion and salt precipitation. The collagen samples were analyzed by SDSPAGE (Figure 1A and B), and collagen chains of the bands were assigned based on the migration patterns.28 Type I collagen is obviously dominant but type III collagen is also presented as about 5−10% of the total. The other types of collagens were not detected by CBB staining. For type I collagen, both α1 and α2 bands from OI fibroblasts appeared as a doublet. The chains of OI type I collagens, which migrated less than those of the normal α chains, are presumably overmodified. A disulfide-bonded dimer of OI-1 appeared under nonreducing conditions, probably due to the substitution of Cys for Gly946 in α1 (I) by a gene mutation.28 Thermal stability of the collagens was also analyzed by circular dichroism (Figure 1C). The denaturation temperatures of OI collagens (39.5 ± 0.4 °C, OI-1; 39.5 ± 0.1 °C, OI-2) were lower than that of normal collagen (41.3 ± 0.5 °C) as previously reported.4,28 The amounts of Hyl derivatives of the collagens were analyzed by amino acid analysis (Table 1). The carbohydrate moieties of GHL/GGHL are labile to acid hydrolysis.32 Thus, Hyl and GHL/GGHL were analyzed

Ol-1 Ol-2

% Hylb

% GHLb

% GGHLb

% total Hylc,d

104.4 ± 16.3 93.1 ± 22.1

155.8 ± 23.3 105.1 ± 25.5

279.0 ± 37.5 176.2 ± 29.7

145.4 ± 16.0 126.6 ± 23.0

a Data represent the mean ± S.D. of three separate experiments. bData were obtained by alkaline hydrolysis. cData were obtained by acid hydrolysis. dTotal Hyl = Hyl + GHL + GGHL.

following alkaline hydrolysis, and total Hyl (Hyl + GHL+ GGHL) were analyzed following acid hydrolysis. From the alkaline-hydrolyzed analysis, Hyl was nearly unchanged, and GHL slightly increased (155.8%, OI-1; 105.1%, OI-2) in both OI collagens compared to the normal collagen. On the other hand, GGHL increased significantly (279.0%, OI-1; 176.2%, OI-2) in the OI collagens, accounting for increases of total Hyl. This particularly significant increase of GGHL in OI collagen is consistent with previous reports.3,4 2227

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Table 2. Results of Comprehensive LC−MS/MS Analysis of O-Glycosylated Peptides of Type I and Type III Collagens Using the Hydrazide Method collagen Type I

chain

positiona

glycosylation site

sequence of identified peptideb

z

m/z

time (min)

α1

76−90 91−108 145−183 145−183 193−237 193−237 265−290 520−555 520−555 586−618 586−618 658−687 658−687 76−90 145−192 145−192 193−237 193−237 520−555 625−651 625−651 655−687 655−687 85−99 325−342

87 99 174 174 219 219 270 531 531 603 603 684 684 87 174 174 219 219 531 648 648 657 657 96 336

R.GLOGTAGLOGMK#GHR.G R.GFSGLDGAK*GDAGPAGPK.G R.GNDGATGAAGPOGPTGPAGPOGFOGAVGAK*GEAGPQGPR.G R.GNDGATGAAGPAGNOGADGQOGAK*GANGAOGIAGAOGFOGAR.G R.GEOGPOGPAGAAGPAGNOGADGQOGAK*GANGAOGIAGAOGFOGAR R.GEOGPOGPAGAAGPAGNOGADGQOGAK#GANGAOGIAGAOGFOGAR.G K.GDTGAK*GDAGAOGAOGSQGAOGLQGMOGER.G R.GANGAOGNDGAK*GDAGAOGAOGSQGAOGLQGMOGER.G R.GANGAOGNDGAK#GDAGAOGAOGSQGAOGLQGMOGER.G R.GLTGPIGPOGPAGAOGDK*GESGPSGPAGPTGAR.G R.GLTGPIGPOGPAGAOGDK#GESGPSGPAGPTGAR.G K.GDAGPOGPAGPAGPOGPIGNVGAOGAK*GAR.G K.GDAGPOGPAGPAGPOGPIGNVGAOGAK#GAR.G R.GFOGTOGLOGFK#GIR.G R.GSDGSVGPVGPAGPIGSAGPOGFOGAOGPK*GEIGAVGNAGPAGPAGPR.G R.GSDGSVGPVGPAGPIGSAGPOGFOGAOGPK#GEIGAVGNAGPAGPAGPR.G R.GEVGLOGLSGPVGPOGNOGANGLTGAK*GAAGLOGVAGAOGLOGPR.G R.GEVGLOGLSGPVGPOGNOGANGLTGAK#GAAGLOGVAGAOGLOGPR.G R.GPSGPOGPDGNK*GEOGVVGAVGTAGPSGPSGLOGER.G R.GEVGPAGPNGFAGPAGAAGQOGAK*GER.G R.GEVGPAGPNGFAGPAGAAGQOGAK#GER.G K.GPK*GENGVVGPTGPVGAAGPAGPNGPOGPAGSR.G K.GPK#GENGVVGPTGPVGAAGPAGPNGPOGPAGSR.G K.GPAGIOGFOGMK#GHR.G R.GPAGPNGIOGEK*GPAGER.G

3 3 4 4 4 4 3 4 4 4 4 3 3 3 4 4 4 4 4 3 3 4 4 4 3

607.62 593.94 894.66 935.18 1015.96 1056.47 852.39 851.36 891.87 762.61 803.13 915.11 969.12 630.30 1074.77 115.28 1035.77 1076.28 848.65 857.73 911.75 757.62 798.13 463.46 618.94

14.5 14.8 15.4 15.4 15.4 15.4 14.3 14.7 14.7 15.4 15.4 15.4 15.0 16.4 16.9 16.9 17.5 17.5 15.6 15.1 15.1 15.3 15.3 15.2 15.1

α2

Type III

α1

The numbering of residues begins with the triple-helical portion of the chains. First residue corresponds to residue 179 of Uniprot # P02452 (type I collagen alpha 1 chain), residue 91 of Uniprot # P08123 (type I collagen alpha 2 chain) and residue 168 of Uniprot # P02461 (type III collagen alpha 1 chain). bO indicates hydroxyproline, K* indicates GHL, and K# indicates GGHL.

a

Identification of GHL/GGHL Peptides Using the Hydrazide Method

between the normal and OI collagens by using the hydrazide method and SILAC.

GHL/GGHL-containing peptides from the normal and OI collagens were purified using the hydrazide method after trypsin digestion.22 The enriched glycopeptides were subjected to LC−MS/MS analysis for identification of the sequence and glycosylation site. Many GHL/GGHL peptides of type I collagen were identified, and a few GHL/GGHL peptides of type III collagen were also identified. The lists of the identified glycopeptides and their modification sites in type I and type III collagens are shown in Table 2. The glycosylation sites were largely located in the N-terminal side of the triple-helical domain, and all of the glycosylated Hyl lay in the Y position of X-Y-Gly triplets. Because GHL/GGHL are not cleaved by trypsin,33,34 the glycopeptides were long with a high charge state (+3 or +4). The identified glycosylation sites included all of the sites identified in our previous study where we analyzed bovine type I collagen using the hydrazide method (Lys87 in both α1 and α2 chains; Lys99 and Lys684 in α1 chain; Lys174, Lys219, Lys648 and Lys657 in α2 chain),22 and several additional ones were also found (Lys531 in both α1 and α2 chains; Lys174, Lys219, Lys270 and Lys603 in α1 chain). The MS/MS spectra of newly identified GHL/GGHL peptides, including from type III collagen, are shown in Figure S1 (Supporting Information). In total, we identified 14 GHL/GGHL sites in human type I collagen (and 2 sites in human type III collagen) from the culture media of normal and OI skin fibroblasts. In the next step, we quantified the relative amounts of the glycopeptides

Quantification of the GHL/GGHL Peptides of OI Collagens

Because the hydrazide method is assumed to be unsuitable for quantitative analysis, we utilized SILAC to ensure quantification precision. OI cells were incubated in labeling media with isotopically heavy Lys and ascorbic acid for 3 days, and normal cells were also incubated in the same conditions, except that Lys was substituted for the heavy Lys. In general, SILAC labeling needs a relatively long-term culture period (approximately 5 days) to fully incorporate the labeled amino acids into proteins. However, a nonlabeled tryptic peptide derived from the labeled OI collagens was not detected by LC−MS with 3 days of labeling (data not shown), probably because collagen is a secretory and rapidly metabolized protein. After purifying the labeled collagens from the culture media, equal amounts of normal and OI collagens were combined. GHL/GGHL peptides were then purified with the hydrazide method and subjected to LC−MS analysis. The monoisotopic XICs of the GHL/GGHL peptides containing Lys174 of α1 (I) are shown in Figure 2 as an example. The peak heights of the glycopeptides were compared between OI-1 and normal. The amounts of GHL peptides were almost equal. In contrast, the GGHL peptide contents were significantly higher in the OI-1 sample. The results of relative quantification including other GHL/ GGHL peptides of type I and type III collagens are summarized in Table 3. The glycopeptides containing Lys270 and Lys531 of α1 (I) and Lys531 of α2 (I) were not quantified because of their low XIC signals. In OI-1, the relative amount of GHL peptide 2228

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of the GGHL peptides increased ranging from 123.2% to 378.4% in the OI collagen. The same tendency was observed in the case of OI-2. This is consistent with the results of amino acid analysis described in Table 1. Especially large increases of GGHL were observed both at the amino acid and the peptide level. In addition, there were no GHL/GGHL peptides detected only in OI collagens. These results indicate that the overglycosylation of type I collagen, probably also of type III collagen, is due to increases of GGHL at specific sites.

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DISCUSSION The function of collagen O-glycosylations has been suggested to be control of fibrillogenesis, cross-linking, and others.6−8 However, the biological significance of the modifications has not been clearly elucidated. In addition, the roles of the overglycosylation of collagen, which has been observed in type I collagens from tissues and cultured fibroblasts from OI patients,1−4,28,35 are also unknown. In this study, we performed site-specific quantitative analysis of the collagen overglycosylation in OI by LC−MS with the hydrazide method and SILAC. We clearly indicated that the overglycosylation in type I collagen from the culture media of OI skin fibroblasts is not random. The overglycosylation reaction proceeds only at specific Lys residues. LC−MS is a powerful tool for comprehensive proteomic analysis, and also for identification and site-specific quantification of the peptide containing PTMs. However, GHL/GGHLcontaining peptides are difficult to detect by LC−MS because of the heterogeneity of the modifications and the ionization suppression in the presence of nonglycosylated peptides.36,37 To this end, we used the hydrazide method to efficiently detect the glycopeptides. With the glycopeptide purification procedure, 14 GHL/GGHL sites in type I collagen from normal and OI skin fibroblasts were identified with conventional LC−MS. Recently, a study has identified 5 GHL/GGHL sites in type I collagen derived from mouse osteoblasts with highly sensitive nano-LC−MS, all of which are consistent with our data.38 This

Figure 2. SILAC-labeled GHL/GGHL peptides of type I collagen from normal and OI fibroblasts. Equal amounts of nonlabeled normal collagen and 13C6-Lys-labeled OI collagen (OI-1) were combined, and subsequently, GHL/GGHL-containing peptides were purified using the hydrazide method after trypsin digestion. The peak heights of the monoisotopic XICs of the GHL/GGHL peptides were quantified by LC−MS analysis. The monoisotopic XICs of the (A) GHL and (B) GGHL peptides of GNDGATGAAGPOGPTGPAGPOGFOGAVGAK*#GEAGPQGPR [α1 (I), O indicates hydroxyproline, and K*# indicates GHL/GGHL at Lys174] of normal (light, solid line) and OI (heavy, dashed line) collagens are shown.

was almost unchanged compared to normal, except for the three peptides which were more than 20% decreased [Lys684 in α1 (I), Lys174 in α2 (I) and Lys336 in α1 (III)]. In contrast, all

Table 3. Relative Quantification of GHL/GGHL Peptides of Type I and Type III Collagens from OI Fibroblasts Compared to Normal Fibrolabsts Using the Hydrazide Method and SILAC (Data represent the mean ± S.D. of three separate experiments)

a

O indicates hydroxyproline, and K in bold with the number indicates the glycosylation site. bPeptides detected with low XIC signals were not quantified. 2229

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methods analyzes were consistent for OI-2. The reason for this discrepancy is unknown, but the tendency was the same: unchanged or slightly increased GHL and significantly increased GGHL in both OI type I collagens. Recently, OI caused by mutations in genes which encode proteins involved in collagen prolyl 3-hydroxylation and collagen chaperone has been reported.40−44 Overmodification has been also observed in some of these OI type I collagens by SDS-PAGE analysis,40,41 and a study has reported remarkable increases of GGHL in collagen from tail tendon, bone, and skin of prolyl 3-hydroxylase 1 null mice by amino acid analysis.45 The overglycosylation in these OI collagens also occurs during delayed triple helix formation caused by the defects of these proteins. Thus, the mechanism of overglycosylation is possibly the same as that of OI caused by a collagen gene mutation. A study has reported that lysyl hydroxylase (LH) isoforms including LH3, which possesses glycosyl transferase activity in addition to LH activity for collagen,9 preferentially bind to specific sequences, but the data have not indicated strict sequence specificity for the enzymes.46 In addition, the enzymatic activity assay has been restricted to Lys hydroxylation. A sequence rule for the glycosylation of Hyl has not been identified. However, the existence of consensus sequences for GHL/GGHL modification can be presumed from the sitespecific overglycosylation of OI type I collagen observed in this study. The glycosylation of specific Hyl may be important for the biological function of the modifications, for example, probable function for cross-link formation at Lys87 of α1 (I).38,47 In addition, 4 GHL/GGHL sites (Lys87, Lys174, Lys219 and Lys531) were identified in both α1 (I) and α2 (I) in this study. This similarity between the chains also suggests that the location of the collagen glycosylations is important for their pathological and physiological roles.

shows the impact of the glycopeptide purification method on the analysis of the collagen O-glycosylations, although there are differences in the origin of the collagens. Although many GHL/GGHL sites were identified in type I collagen from normal and OI fibroblasts with this method, the mechanism by which glycosylation increases in OI collagen remained uncertain. To answer this question, we utilized SILAC, which compensates for the quantitative weakness of the hydrazide method. The site-specific quantitative analysis revealed two things. (i) GGHL increased significantly in all of the analyzed sites of OI type I collagen, while GHL did not increase. (ii) There was no glycosylation site exclusively detected in the OI collagen. All quantified glycopeptides were also detected in normal collagen, albeit with relatively low signals. Taken together, these data indicate that the glycosylation reaction proceeds at specific sites from Lys/Hyl to GHL and subsequently GGHL during the delayed triple helix formation in OI type I collagen, and therefore, GGHL accumulates at the sites as an end product. This overglycosylation model of type I collagen in OI is illustrated in Figure 3. Overmodification of type III collagen in OI has also

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-297-71-3046. Fax: +81-297-71-3041. E-mail: [email protected]. Figure 3. Overglycosylation at specific sites of type I collagen in OI. (A) Specific Lys residues are post-translationally modified in the endoplasmic reticulum until collagen folds into a triple-helical conformation. Lysyl hydroxylases add a hydroxyl group to the Lys, and glycosyltransferases add galactose and subsequent glucose to the Hyl. The O-glycosylation sites exist as mixtures of Lys, Hyl, GHL and GGHL in general. (B) Mutation in the type I collagen gene in OI delays the triple helix formation leading to prolonged exposure time to all the enzymes. Consequently, the Hyl and GHL contents are nearly unchanged in a series of modification reactions, and in contrast, GGHL accumulates as an end product at the specific glycosylation sites.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Kazunori Mizuno at Shriners Hospital for Children for his helpful comments on the manuscript. ABBREVIATIONS OI, osteogenesis imperfecta; Hyl, hydroxylysine; GHL, galactosyl-hydroxylysine; GGHL, glucosyl-galactosyl-hydroxylysine; HRP, horseradish peroxidase; SILAC, stable isotope labeling by amino acids in cell culture; PTMs, post-translational modifications; MRM, multiple reaction monitoring; XICs, extracted ion chromatograms; LH, lysyl hydroxylase

been reported previously.1,39 From our data, it appears that overglycosylation of type III collagen in OI occurs in the same manner as that of type I collagen, although only two glycosylation sites were analyzed in type III collagen. The results of quantification of GHL in OI-1 differed between amino acid analysis (increased) and glycopeptide analysis (unchanged or decreased), while the results for these

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