Cyclophilin B deficiency causes abnormal dentin collagen matrix

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Cyclophilin B deficiency causes abnormal dentin collagen matrix Masahiko Terajima, Yuki Taga, Wayne A. Cabral, Masako Nagasawa, Noriko Sumida, Shunji Hattori, Joan C. Marini, and Mitsuo Yamauchi J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00190 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 16, 2017

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Journal of Proteome Research

Cyclophilin B deficiency causes abnormal dentin collagen matrix Masahiko Terajima a,#, Yuki Taga b,#, Wayne A. Cabral c, Masako Nagasawa d, Noriko Sumida a, Shunji Hattori b, Joan C. Marini c, and Mitsuo Yamauchi a,* a

Oral and Craniofacial Health Sciences, School of Dentistry, University of North Carolina, Chapel Hill, North Carolina 27599, USA b

c

Nippi Research Institute of Biomatrix, Ibaraki 302-0017, Japan

Section on Heritable Disorders of Bone and Extracellular Matrix, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA d

Division of Bio-Prosthodontics, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan

ABSTRACT Cyclophilin B (CypB) is an endoplasmic reticulum-resident protein that regulates collagen folding, and also contributes to prolyl 3-hydroxylation (P3H) and lysine (Lys) hydroxylation of collagen. In this study, we characterized dentin type I collagen in CypB null (KO) mice, a model of recessive osteogenesis imperfecta type IX, and compared to those of wild-type (WT) and

ACS Paragon Plus Environment

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heterozygous (Het) mice. Mass spectrometric analysis demonstrated that the extent of P3H in KO collagen was significantly diminished compared to WT/Het. Lys hydroxylation in KO was significantly diminished at the helical cross-linking sites, α1/α2(I) Lys-87 and α1(I) Lys-930, leading to a significant increase in the under-hydroxylated cross-links and a decrease in fully hydroxylated cross-links. The extent of glycosylation of hydroxylysine residues was, except α1(I) Lys-87, generally higher in KO than WT/Het. Some of these molecular phenotypes were distinct from other KO tissues reported previously, indicating the dentin-specific control mechanism through CypB. Histological analysis revealed that the width of predentin was greater and irregular, and collagen fibrils were sparse and significantly smaller in KO than WT/Het. These results indicate a critical role of CypB in dentin matrix formation, suggesting a possible association between recessive osteogenesis imperfecta and dentin defects that have not been clinically detected.

Keywords:

Cyclophilin

B,

Collagen,

Post-translational

modification,

Hydroxylysine,

Glycosylation, Dentin.

INTRODUCTION Collagens comprise a large family of structurally related extracellular matrix proteins distributed throughout the body.1 Among all of the genetic types of collagen identified, type I collagen is the most abundant type and is the major organic matrix component of most connective tissues, including bone, dentin, skin, and tendon. It is a heterotrimeric molecule composed of two α1(I) and one α2(I) chains forming a long uninterrupted triple helix with short


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non-helical domains at both N- and C-termini (telopeptides). One of the functionally important characteristics of collagen is its unique, sequential post-translational modifications of lysine (Lys) residues, including hydroxylation forming 5-hydroxylysine (Hyl) in both helical and telopeptide domains, mono- and di-glycosylation of Hyl in the helical domain, oxidative deamination of Lys and Hyl in the telopeptides and subsequent covalent intermolecular crosslinking.2 The importance of these post-translational modifications cannot be overemphasized as they are critical for collagen fibrillogenesis, stability and mineralization.3-7 The biological importance of these modifications is also evidenced by a series of recent studies on recessive osteogenesis imperfecta (OI) showing that defects in genes encoding Lys modifying enzymes cause brittle bones and other connective tissue diseases.8, 9 These studies also provide an important insight into the molecular mechanism by which Lys hydroxylation, the first step of a series of Lys modifications, is regulated by other endoplasmic reticulum (ER) resident chaperones and foldases that interact with lysyl hydroxylases. Cyclophilin B (CypB), encoded by the PPIB gene, is an ER-resident peptidyl-prolyl cis-trans isomerase and a component of the prolyl 3-hydroxylase complex, thus, playing critical roles in collagen folding and prolyl 3-hydroxylation (P3H). Mutations in PPIB cause recessive OI type IX.10, 11 Our recent studies on CypB null mice, a model of recessive OI, demonstrated that CypB deficiency affects not only P3H but also Lys modifications of type I collagen, i.e. Lys hydroxylation, glycosylation of Hyl and intermolecular cross-linking. This resulted in abnormal fibrillogenesis and tissue formation in bone, skin and tendon.12,

13

However, the extent of

molecular and tissue phenotypes appears to be tissue-specific.


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Though it is well documented that dentinogenesis imperfecta (type I) is associated with autosomal dominant OI,14 its association with recessive OI caused by defective collagen posttranslational modification has not been clinically detected.9 In this study, we performed in-depth analysis of type I collagen in Ppib-/- (CypB KO, hereafter) mouse dentin using LC-MS and cross-link analysis and histological evaluation to gain insight into the potential role of CypB-associated type I collagen post-translational modifications in dentin matrix formation.

EXPERIMENTAL SECTION Ppib Null Mice. Animal care and experiments were performed in accordance with a protocol approved by the NICHD ACUC committee. Ppib null mice have recently been generated as a mouse model of recessive OI12 and, in this model, Ppib transcripts and CypB protein were not detected in primary cells and tissues. Collagen Preparation for Biochemical Analysis. Erupted portions of mandibular incisors were dissected by a razor blade from 2 month old WT, Het, and CypB KO mice. Extra care was taken to avoid contamination with surrounding gingiva, skin and bone. Though enamel was not separated from the specimens, a small amount of enamel proteins devoid of collagen would not affect the following studies as we used the 4-hydroxyproline (Hyp) values to normalize for all, except Pro hydroxylation, biochemical analyses and type I collagen derived peptides for mass spectrometric analysis. All operations were carried out on ice at 4°C. The dissected incisors were pulverized with a pestle and mortar to a fine powder under liquid nitrogen. Pulverized samples were washed several times with cold phosphate-buffered saline (PBS), and cold distilled water by repeated centrifugation at 4,000×g for 30 min, and lyophilized. Teeth powder was then


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demineralized with 0.5 M ethylenediamine-tetraacetic acid (EDTA), pH 7.5, for 2 weeks with several changes of EDTA solution at 4,000×g. The EDTA-insoluble residue was thoroughly washed with cold distilled water by repeated centrifugation at 4,000×g and lyophilized. Protease Digestion of Tooth Samples. Demineralized dentin collagen from WT, Het, and KO mice were digested with trypsin or collagenase/pepsin to analyze the Lys post-translational modifications at specific molecular sites within the triple helical domain or telopeptides of type I collagen, respectively, by following procedures slightly modified from a previously reported method.15 Briefly, the demineralized samples were heated at 65°C for 15 min in 200 mM ammonium bicarbonate and digested with trypsin (Promega, Madison, WI; 1% w/w) at 37°C for 16 hours. The samples were again heated at 65°C for 10 min and digested with trypsin (0.5% w/w) at 37°C for 3 hours.15,

16

This repeated denaturation/digestion method effectively

solubilizes dentin collagen. In parallel, the samples were also subjected to collagenase digestion using recombinant collagenase from Grimontia hollisae (Wako Chemicals, Osaka, Japan)17 in 100 mM Tris-HCl/5 mM CaCl2 (pH 7.5). After centrifugation, filtration with a 0.45 µm membrane, and addition of acetic acid (final 0.5 M), the collagenase-digests were further treated with pepsin (Sigma-Aldrich, St. Louis, MO; 2% w/w) at 37°C for 16 hours. The enzyme was deactivated by heating at 100°C for 5 min. LC-Triple Quadrupole (QqQ)-MS. The peptide solutions digested with trypsin were subjected to LC-MS using a hybrid QqQ/linear ion trap 3200 QTRAP mass spectrometer (AB Sciex, Foster City, CA) coupled to an Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA). Peptides were separated on an Ascentis Express C18 HPLC Column (5 µm particle size, L×I.D. 150 mm×2.1 mm; Supelco, Bellefonte, PA) at a flow rate of 500 µl/min with a binary gradient as follows: 98% solvent A (0.1% formic acid) for 2.5 min, linear gradient


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of 2–50% solvent B (100% acetonitrile) for 12.5 min, 90% solvent B for 2.5 min, and 98% solvent A for 2.5 min. The separated peptides were ionized and detected in positive ion mode with data acquisition using Analyst software 1.6.2 (AB Sciex). The MS scan and MS/MS acquisition were performed over the m/z ranges of 400–1300 and 100–1700, respectively. MS/MS fragmentation was performed by information–dependent acquisition method that was operated by selecting the two most intense precursor ions of the prior survey MS scan (intensity threshold, 100000 cps; charge state, +2 to +4). The collision energy was automatically determined based on the mass and charge state of the precursor ions using rolling collision energy. Site occupancy of P3H and Lys hydroxylation/ glycosylation (Lys, Hyl, galactosylhydroxylysine (G-Hyl), and glucosyl-galactosyl-hydroxylysine (GG-Hyl)) were calculated using the ratio of peak areas of peptides containing the respective molecular species as reported previously.12 LC-Quadrupole Time-of-Flight (QTOF)-MS. The peptide solutions digested with trypsin or collagenase/pepsin were subjected to LC-MS using a maXis II QTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled to a Shimadzu Prominence UFLC-XR system (Shimadzu, Kyoto, Japan). Peptide separation was performed using the Ascentis Express C18 HPLC Column under the same conditions as the LC-QqQ-MS. The separated peptides were ionized and detected in positive ion mode with data acquisition using otofControl version 4.0 (Bruker Daltonics). The MS scan and MS/MS acquisition were performed over the m/z ranges of 50–2500 with a frequency of 2 Hz (peptide quantification) or 5 Hz (peptide identification). MS/MS fragmentation was performed in the MS/MS mode (4 precursor ions; intensity threshold, 1500 cts). The collision energy was automatically determined based on the mass and charge state of the precursor ions with the default setting.


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Peptide identification was performed by searching the MS/MS data converted to .mgf files against the UniProtKB/Swiss-Pilot database (release 2014_08) for Mus musculus species (16676 protein entries) using ProteinPilot software 4.0 (AB Sciex) with the ParagonTM algorithm.18 We have deposited the MS datasets to the ProteomeXchange consortium via the jPOST partner repository with the dataset identifier PXD006021.19, 20 Search parameters included digestion by collagenase (manually defined as peptide bond cleavage before glycine), biological modifications ID focus, and 95% protein confidence threshold. Search criteria of posttranslational modifications were optimized for collagen analysis as described previously. 21 We defined the confidence threshold of the identified peptides to be 95%. Amino Acid and Cross-link Analyses. Demineralized samples (~1.0 mg each) were reduced with standardized NaB3H4 as reported.22, 23 The reduced samples were washed with cold distilled water several times by repeated centrifugation at 4,000×g and lyophilized. Reduced collagen was hydrolyzed with 6 N HCl and subjected to amino acid analysis as described previously.24 The extent of Lys hydroxylation in collagen was calculated as Hyl/collagen based on the value of 300 residues of Hyp/collagen. Proline (Pro) hydroxylation was calculated by 4-Hyp/(Pro+4Hyp). Aliquots of the hydrolysates were also subjected to cross-link analysis by using Radiomatics Flo-one/Beta

software

(Packard

dehydrodihydroxylysinonorleucine

BioScience,

Meriden,

(dehydro-DHLNL)/its

CT).

Upon ketoamine

reduction, and

dehydrohydroxylysinonorleucine (dehydro-HLNL)/its ketoamine are reduced to stable secondary amines, DHLNL and HLNL. The reducible cross-links were analyzed as their reduced forms (i.e. DHLNL and HLNL). A cross-link precursor, hydroxylysine-aldehyde (Hylald), was also identified and quantified as its reduced form, dihydroxynorleucine (DHNL). Hereafter, the terms


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DHNL, DHLNL and HLNL will be used for both the unreduced and reduced forms. The levels of Hylald, immature reducible cross-links (DHLNL and HLNL) and mature non-reducible crosslinks, pyridinoline (Pyr) and deoxy-Pyr (d-Pyr), were quantified as mol/mol of collagen.24 Histological Evaluation. WT, Het and KO mouse mandibles including the first molars were dissected and subjected to histological evaluation (3 mandibles/group). The specimens were immersed for 3 days with 10% formalin and demineralized with 0.5 M of EDTA (pH 7.4) for 2 weeks, immersed in 70% ethyl alcohol, dehydrated through ascending gradations of ethanol, embedded in paraffin, and sectioned into 5 µm thick sections. After hydration, the slices were stained with hematoxylin and eosin (H&E) and observed under light microscopy (Olympus BX40; Olympus, Tokyo, Japan). One hundred predentin sections in the region of pulp horn were randomly selected and the diameters were measured using ImageJ version 1.44p software (National Institutes of Health, Bethesda, MD). The collagen fibrils were characterized by transmission electron microscopy (TEM). The specimens were fixed with 2.5% EM grade glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 3 days, and demineralized with 0.5 M EDTA (pH 7.4) for 2 weeks. The samples were then postfixed in potassium ferrocyanide-reduced osmium for 1 hour at room temperature. After rinsing with distilled water, the samples were dehydrated with a graded series of ethanol concentrations and embedded in PolyBed-812 epoxy resin (Polysciences, Warrington, PA). Seventy nm-thick sections were cut, mounted on copper Formvar-carbon filmed grids, and stained with 4% urinary acetate and Reynolds’ lead citrate.25 Cross-sectional and longitudinal views of the collagen fibrils in predentin were observed using a LEO EM-910 transmission electron microscope operating at 80 kV (Carl Zeiss SMT, Peabody, MA), and images were taken at ×5,000 using a Gatan Orius SC1000 CCD camera with Digital Micrograph 3.11.0 (Gatan, Inc.,


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Pleasanton, CA). Seven hundred fibrils in predentin in the region of pulp horn were randomly selected and the diameters were measured using ImageJ version 1.44p software as stated above. Statistical Analysis—Statistical analyses were performed using Jmp®8.0 software (SAS Institute Inc., Cary, NC). Statistical differences were determined by Kruskal-Wallis one-way analysis of variance and means comparisons by Student’s t test. The data were presented as means ± S.D. and a p value less than 0.05 was considered significant.

RESULTS Post-translational Modifications. The amino acid compositions in WT, Het and KO are indicated in Table S1. The extents of Lys and Pro hydroxylations are shown in Table 1. Lys hydroxylation showed no statistical difference among the three groups indicating that absence of CypB did not affect the overall level of Lys hydroxylation in collagen. The 4-Pro hydroxylation calculated as 4-Hyp/(Pro+4-Hyp) was 0.42-0.43 in all of the groups suggesting that this modification was also not affected by the absence of CypB, which is consistent with bone, skin and tendon.12, 13 However, the effect on dentin Lys hydroxylation is distinct from these tissues. In CypB KO mouse skin and tendon collagen, Lys hydroxylation was decreased as compared with those from WT/Het mice, respectively,12, 13 whereas CypB KO bone showed a slight increase.12 Collagen Type. Type I and III collagens with negligible amounts of other types (VI, XI and XII) were identified by LC-QTOF-MS/MS analysis of tryptic digests of tooth samples in all WT, Het and KO mice (Table S2). The type I/III ratio was roughly estimated based on a number of identified peptides with >95% confidence. The data indicated that the majority of collagen was type I collagen (>90%) in all three groups. Type III collagen was identified as a minute collagenous component, which is similar to tail tendon.13


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Alterations of Collagen Post-translational Modifications at Specific Molecular Loci. The distributions of Pro and Lys modifications at specific sites within the triple helical region of type I collagen were semi-quantitatively estimated by LC-QqQ-MS analysis using tryptic digests of tooth samples as described in previous studies12, 13 (Table 2). The values of WT and Het were almost identical to each other with no statistical difference (p>0.1 at all sites). The extent of P3H at α1(I) Pro-986 was markedly diminished by the absence of CypB (95.4% for WT, 96.4% for Het, and 3.1% for KO) (Table 2A), consistent with our previous reports for bone, skin, and tendon.12, 13 Though to a much lesser extent, P3H at α1(I) Pro-707 was also decreased in KO (17.9% for WT, 17.9% for Het, and 10.7% for KO) whereas a decrease in P3H was not observed for α2(I) Pro-707. Lys modifications within the triple helical region of type I collagen were affected in a site-specific manner. By evaluating the ratio of Lys to total Hyl (glycosylated and non-glycosylated Hyl), significant underhydroxylation was observed in KO only at the helical cross-linking sites (Figure 1A), α1 Lys-87, at which 10.2% for WT, 8.6% for Het, and 32.0% for KO, and α2 Lys-87, at which 20.6% for WT, 24.3% for Het, and 45.7% for KO of Lys residues were not hydroxylated (Figure 1B and C, Table 2A). Collagenase/pepsin digestion for telopeptide analysis described below identified a peptide containing α1(I) Lys-930, another helical cross-linking site, similar to a recent study using bacterial collagenase (Figure S1 and Table

S3).26

Both

Lys

residues

in

the

peptide

containing

α1(I)

Lys-918/930

(GDKGETGEQGDRGIKGHR) in WT/Het were almost fully hydroxylated (Lys+Lys = 2.2%, Lys+Hyl=9.5-9.7%), but those in KO, 17.8% was non-hydroxylated indicating that α1(I) Lys930 in KO was at least ~15% less hydroxylated than WT/Het (Figure 1D, Table 2). The Lys hydroxylations at other sites examined, i.e. α1 Lys-174, α1 Lys-219, α1 Lys-603, α2 Lys-174, and α2 Lys-219 were not affected in CypB KO type I collagen (Table 2A) indicating that the


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alteration occurred only at specific cross-linking sites. Since the helical cross-linking Lys/Hyl residues represent only ~5% of the total Lys residues in type I collagen,24 the extent of alterations at these sites in KO collagen did not significantly affect the total Lys hydroxylation per collagen. Furthermore, four glycosylation sites, α1 Lys-87, α1 Lys-174, α2 Lys-174, and α2 Lys-219, were identified. The effect of CypB KO on glycosylation was found to be altered in a sitespecific manner. When calculated as percentages of GG-, G-, and free-Hyl in total Hyl (Table 2B), the relative abundance of G-Hyl at α1-87, the major glycosylation site, was significantly lower in CypB KO collagen compared to that of Het, while free- and GG-Hyl at this site showed no difference. No glycosylation was detected at α2-87, which is consistent with our previous reports.12 At other sites in CypB KO, i.e. α1-174, α2-174, and α2-219, the relative abundance of GG-Hyl was significantly higher than those of WT and Het. In addition, G-Hyl at α1-174 and α2-219 also showed significantly higher level compared to WT and Het. Lys hydroxylation in the N- and C-telopeptides of type I collagen were analyzed by LCQTOF-MS after sequential digestion by Grimontia collagenase and pepsin. We previously analyzed Lys hydroxylation in the telopeptides using the Grimontia collagenase.13 However, collagenase-digested α1(I) C-telopeptide still contained Hyp at its N-terminus, which hampered simple evaluation of Lys hydroxylation without MS/MS fragmentation. Thus, we performed further digestion with pepsin to cleave GPPSGGYDF-SFLPQPPQEKSQDGGR bond in the Ctelopeptide. Both in the α1(I) N-telopeptide (GYDEKSAGVSVP) and α1(I) C-telopeptide (SFLPQPPQEKSQDGGR) (Figure S2), there was no significant difference in Lys hydroxylation between KO and WT/Het (Table 2A).


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Collagen Cross-link Analysis. Figure 2 shows the results of cross-link analysis obtained from the acid hydrolysates of reduced WT, Het, and KO dentin collagen. In all three groups, a crosslink precursor aldehyde, DHNL, reducible, immature bifunctional cross-links, DHLNL and HLNL, and mature trifunctional cross-links, Pyr and d-Pyr, were identified. WT and Het showed essentially identical cross-linking pattern with no statistical difference. In KO, when compared to WT/Het, however, there were significant differences: increases in DHNL (p

Cyclophilin B deficiency causes abnormal dentin collagen matrix

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