Abstract An N-glycomic analysis of plasma proteins was performed in Japanese semisupercentenarians (SSCs) (mean 106.7 years), aged controls (mean 71.6 years), and young controls (mean 30.2 years) by liquid chromatography/mass spectrometry (LC/MS) using a graphitized carbon column. Characteristic N-glycans in SSCs were discriminated using a multivariate analysis; orthogonal projections to latent structures (O-PLS). The results obtained showed that multi-branched and highly sialylated N-glycans as well as agalacto- and/or bisecting N-glycans were increased in SSCs, while biantennary N-glycans were decreased. Since multi-branched and highly sialylated N-glycans have been implicated in anti-inflammatory activities, these changes may play a role in the enhanced chronic inflammation observed in SSCs. The levels of inflammatory proteins, such as CRP, adiponectin, IL-6, and TNF-α, were elevated in SSCs. These results suggested that responses to inflammation may play an important role in extreme longevity and healthy aging in humans. This is the first study to show that the N-glycans of plasma proteins were associated with extreme longevity and healthy aging in humans.

Citation: Miura Y, Hashii N, Tsumoto H, Takakura D, Ohta Y, Abe Y, et al. (2015) Change in N-Glycosylation of Plasma Proteins in Japanese Semisupercentenarians. PLoS ONE 10(11): e0142645. https://doi.org/10.1371/journal.pone.0142645 Editor: Jon M. Jacobs, Pacific Northwest National Laboratory, UNITED STATES Received: August 8, 2015; Accepted: October 23, 2015; Published: November 11, 2015 Copyright: © 2015 Miura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: The Japan Society for the Promotion of Science (No. 24659141 to TE) (http://www.jsps.go.jp/j-grantsinaid/index.html), and Mitsui Sumitomo Insurance Welfare Foundation (No. 18 to YM)(http://www.ms-ins.com/welfare/): The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: SSC, semisupercentenarian; O-PLS, orthogonal projections to latent structures; PCA, principle component analyses; LC/MSn, Liquid chromatography/multiple stage mass spectrometry; MVA, multivariate analysis; CRP, C-reactive protein; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; Con A, concanavalin A

Introduction Aging is not caused by a single factor or process; it is modulated by various genetic and environmental factors such as oxidative stress and lifestyle. However, the mechanisms underlying the aging process currently remain unclear [1]. Semisupercentenarians (SSCs; older than 105 years) are regarded as a model of human longevity because they have aged successfully [2]. Therefore, analyses of SSCs are expected to assist in elucidating the human aging process. We have physiologically and biochemically analyzed SSCs, and demonstrated that the content of oxidative stress-related proteins in the plasma of SSCs was altered from a young age [3]. These findings suggest that the regulatory mechanisms underlying oxidative stress are important for human longevity and healthy aging. Since post-translational modifications to proteins do not occur according to a genetic template, they are sensitive to various biological changes. Typical modifications to proteins, such as phosphorylation, glycosylation, and acetylation, depend on the activities of enzymes such as kinases, glycosyltransferases, and acetylases, respectively. Since the aging process is modulated by environmental factors, e.g. oxidative stress, heat shock, etc [1], a focus on age-dependent alterations in post-translational modifications in SSCs is considered important for obtaining a deeper understanding of human longevity and healthy aging. Glycosylation is one of the most common post-translational modifications to proteins. Recent studies suggest that glycans modify the functions of proteins and play important roles in various biological processes such as molecular recognition, cell adhesion, and immunological defense systems. The biosynthesis of glycans is known to depend on the activities of glycosyltransferases (transfer of sugars) and glycosidases (hydrolysis of sugars), and their activities are easily altered by changes in the physiological conditions of cells. Therefore, although glycans are highly susceptible to changes in the biological environment such as those caused by various disorders [4–7] and aging [8], the mechanisms responsible have not yet been examined in detail. N-linked glycosylation (N-glycan), which is covalently conjugated to asparagine residues in a consensus sequence (Asn-X-Ser/Thr), is synthesized in the endoplasmic reticulum, in which a lipid-linked precursor oligosaccharide is attached to a protein, followed by the concerted action of glycosyltransferases in the Golgi apparatus. More than half of mammalian proteins are estimated to be glycosylated. Human plasma proteins, except for albumin and CRP (C-reactive protein), are mostly modified by glycans. Furthermore, since plasma proteins are derived from various tissues and organs, their properties are affected by the physiological or pathological conditions of various tissues and organs, indicating that plasma proteins and their glycans are good targets for monitoring healthy conditions. N-glycan structures have a number of positional isomers and anomeric configurations including branching; therefore, they are very diverse. As a result, difficulties have been associated with obtaining N-glycan structural information until recently. Among several analytical methods, mass spectrometry (MS) is currently the most efficient and promising analytical tool for elucidating N-glycan structures. N-Glycan analyses have often been performed using liquid chromatography (LC) and capillary electrophoresis in combination with MS [9, 10]. These methods are well established and regarded as platform technologies for N-glycan profiling; however, manually distinguishing complex and unclear differences between the N-glycan heterogeneities of samples when significant quantitative and qualitative changes are not observed is very challenging. We applied previously orthogonal projections to latent structures (O-PLS) in order to characterize the N-glycan heterogeneities of erythropoietin using the peak area ratios of N-glycans in mass spectra obtained by LC/MS [11]. Differences in N-glycan heterogeneities were visualized and digitalized using these methods, and characteristic N-glycans were successfully identified. Previous studies reported the application of O-PLS to the search for biomarkers in proteomics [12–15]. In the present study, we performed a glycomic analysis of N-glycan in the plasma proteins of SSCs using LC/MS and O-PLS, and demonstrated the characteristic structures of N-glycans in SSCs, which may play a role in extreme longevity and successful aging in humans.

Materials and Methods Subjects Six female semisupercentenarians (SSCs, mean age 106.7 ± 0.5 years) were recruited in this study. None were in an acute care situation or receiving tube feeding. The disease histories of these SSCs included coronary artery disease, stroke, diabetes, hypertension, and cancer. Five female aged subjects (mean age 71.6 ± 1.5 years) and 5 female young subjects (mean age 30.2 ± 8.1 years) were recruited as healthy volunteers. The young subjects were free from diseases and had no relevant history, such as coronary artery disease, stroke, diabetes, hypertension, and cancer. The aged subjects were also free from diseases, but had histories of coronary artery disease and hypertension. All subjects enrolled in this study were Japanese. Twenty milliliters of non-fasting venous blood was collected, and plasma was immediately separated by centrifugation at 4°C and stored at -80°C until subsequent assays. This study was approved by the Ethics Committees of the Tokyo Metropolitan Institute of Gerontology (approval number 1). All participants in this study gave written informed consent to participate. Materials N-Glycosidase F was purchased from Roche Diagnostics (Mannheim, Germany). Guanidine hydrochloride was purchased from Nacalai Tesque (Kyoto, Japan). Dithiothreitol and monoiodoacetate were purchased from Sigma (St. Louis, MO, USA). All other reagents were of the highest quality available. Sample Preparation for N-glycan Profile Analyses Plasma (3 μl) was dissolved in 8 M guanidine hydrochloride / Tris-HCl (pH 8.6). The mixture was reduced by the addition of dithiothreitol for 30 min at 65°C, followed by alkylation with sodium monoiodoacetate for 40 min at room temperature in the dark. The resulting mixture was applied to a PD-10 column (GE Healthcare, Little Chalfont, UK) to remove the reagents, and a fraction of the carboxymethylated proteins was dried. The sample was re-dissolved in 50 mM sodium phosphate buffer containing 10 mM EDTA (pH 8.0). After protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), 5 U of peptide N-glycosidase F, which cleaves between the innermost GlcNAc and asparagine residues of N-linked glycoproteins, was added to the sample (100 μg protein) and incubated for 16 h at 37°C. Proteins were removed by precipitation with ice cold methanol (60%) and centrifugation (4°C, 8,000 g x 5 min), and the supernatant including N-glycan was evaporated. N-glycans were reduced in 500 μl of 0.5 M sodium borohydride at room temperature for 16 h and neutralized with acetic acid. Reduced N-glycans were recovered with a solid-phase extraction cartridge (EnviCarb C, Supelco, Bellefonte, PA, USA), lyophilized, and re-dissolved in 25 μl of ultrapure water. Liquid Chromatography/Multiple Stage Mass Spectrometry (LC/MSn) Analyses of sodium borohydride-reduced N-glycans were performed using liquid chromatography/multiple-stage mass spectrometry (LC/MSn). Chromatographic separation was performed using an UltiMate 3000 RSLCnano LC system (Thermo Fisher Scientific, San Jose, CA, USA) with a graphitized carbon column (Hypercarb, 0.1 x 150 mm, 5 μm; Thermo Fisher Scientific). The mobile phase was 5 mM ammonium bicarbonate containing 2% acetonitrile (buffer A) and 5 mM ammonium bicarbonate containing 80% acetonitrile (buffer B). N-glycans were separated at a flow rate of 500 nl/min with a linear gradient of 15%–90% buffer B for 60 min. A mass spectrometric analysis of N-glycans was performed using Fourier transform ion cyclotron resonance linear and ion trap type mass spectrometers (FTMS/ITMS, LTQ-FT, Thermo Fisher Scientific). The analytical conditions were as follows: full mass scan using FTMS (m/z 700–2,000) and data-dependent MS/MS, MS/MS/MS, and MS/MS/MS/MS (MSn) using ITMS; electrospray voltage in positive and negative ion modes, 2.5 and -2.5 kV, respectively; capillary temperature, 200°C; collision energy for MSn experiments, 35%; maximum injection times for FTMS and MSn, 1,250 and 200 ms, respectively; FTMS resolution, 100,000. The peak areas of N-glycans were measured using the Thermo Xcalibur 2.2 SP1.48 Qual Browser (Thermo Fisher Scientific). Multivariate Analysis (MVA) The peak area ratios of each N-glycan to the total peak area of N-glycans were used to perform a principal component analysis (PCA) and orthogonal projections to latent structures (O-PLS) using the MVA software SIMCA-P+ 12.0.1 (Umetrics, Umea, Sweden). Differences in N-glycans between generations were visualized in the score plot obtained by PCA, and the characteristic N-glycans of each subject were found in the loading plot by O-PLS. Contents of N-Acetylneuraminic Acid (NeuNAc) in Plasma N-Glycans Plasma samples at the age of 70 years were obtained from SONIC (Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians). Samples were prepared from 6 μl plasma according to the same method as the N-glycan profile analyses. N-Acetylneuraminic acid was released from N-glycans by the incubation with Arthrobacter ureafaciens sialidase (Nacalai Tesque) in 0.5 M ammonium acetate buffer (pH 5.0) at 37°C for 2 h. The contents of the resulting free N-acetylneuraminic acid were measured using a sialic acid (NANA) fluorometric assay kit (BioVision Inc., Milpitas, CA, USA) according to the manufacturer’s instructions, followed by detection and analyses using the EnVision Multilabel Reader (PerkinElmer Inc., Waltham, MA, USA). Statistics A one-way analysis of variance (ANOVA) (IBM SPSS Statistics version 20.0; IBM, Armonk, NY, USA) was used for statistical analyses. Multiple comparisons were performed by the two-sided Dunnett method. Results were considered significant at a p value of < 0.05. In the case of inflammatory parameters such as CRP, adiponectin, IL-6, and TNF-α, data were logarithmically transformed before statistical analyses.

Discussion In the present study, we investigated longevity-associated N-glycans in SSCs. We performed an N-glycomic analysis of plasma proteins in SSCs, a model of human longevity, using LC/MS and O-PLS. The results obtained showed that multi-branched and highly sialylated N-glycans as well as agalacto- and/or bisecting N-glycans were higher, whereas biantennary N-glycans were lower in SSCs than in young and aged controls. This is the first study to demonstrate that N-glycans are associated with extreme longevity and healthy aging in humans. The effects of aging on N-glycan profiles in human plasma or serum were recently examined using several methods: DNA sequencer-assisted, fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) [16–20], nano-HPLC-chip-TOF-MS [21, 22], UPLC and MALDI-TOF-MS [23], and hydrophilic interaction high performance liquid chromatography (HILIC) [6, 24–26]. All studies found age-dependent increases in non-galactosylated biantennary N-glycans and corresponding decreases in digalactosylated biantennary N-glycans [16, 20, 24, 25]. Additionally, specific glycoproteins, such as IgG, instead of total plasma glycoproteins were reported to show similar alterations with aging [27, 28]. In the present study on SSCs, a reduction was also observed in digalactosyl biantennary N-glycan (No. 15 in Fig 3), suggesting that decrease in the N-glycan is age-associated changes. On the other hand, the increases in multi-branched and highly sialylated N-glycans (No. 1, 2, 6–12 in Fig 3) observed in SSCs were not reported in previous studies on aging. Therefore, these changes may be associated with extreme longevity. Previous studies demonstrated that multi-branched and highly sialylated N-glycans were elevated in response to inflammatory diseases, such as rheumatoid arthritis [29], ulcerative colitis [30], and chronic pancreatitis [31]. However, no subjects in the present study had or had previous histories of inflammatory diseases. On the other hand, chronic inflammation is known to be enhanced with healthy aging [32], and we found that inflammatory factors such as CRP, adiponectin, TNF-α, and IL-6 were elevated in SSCs (Table 2). Therefore, multi-branched and highly sialylated N-glycans may be increased in SSCs as a response to chronic inflammation. The structures of sialic acids, sialyl linkages (α2,3 or α2,6), and O-acetylated forms [33–35] need to be considered because their differences may be relevant to the biological role of plasma glycoproteins in inflammation. In the Leiden Longevity study, Ruhaak et al. examined longevity-associated N-glycans and reported that non-fucosylated biantennary glycans were significantly higher in the offspring of nonagenarians (90 years old) than in controls [25]. However, our results obtained from SSCs were not consistent with these findings. A difference may exist between nonagenarians and SSCs. Nevertheless, a follow-up of individual offspring is awaited in order to determine whether they will also have long lifespans. This information is needed in order to establish whether the N-glycans described above are associated with longevity. No significant feature of fucosylated glycans was observed in the present study. The fucosylation of N-glycans has been implicated in several diseases including cancer [36]. Since the subjects who participated in this study were healthy individuals, fucosylated N-glycans in SSCs may not have been altered. Human longevity may be associated with genes [1]. Several studies have attempted to discover the genetic basis of extreme longevity [37, 38]. A genome-wide association study (GWAS) of N-glycomes in human plasma revealed polymorphisms in several glycogenes [39]; one was MGAT5, which codes for the enzyme involved in the generation of multi-branched N-glycans, while another was SLC9A9, which codes for a proton pump affecting Golgi pH linked to the sialylation of glycans. Further studies are warranted in order to determine whether these genes show any polymorphisms in SSCs, the findings of which may contribute to a better understanding of the roles of glycans in human longevity. In conclusion, we herein identified an enhancement in multi-branched and highly sialylated N-glycans in SSCs. These N-glycan changes may play a role in anti-inflammatory responses against enhanced chronic inflammation. Thus, proper responses to inflammation through N-glycan modulations may be key for longevity and healthy aging in human. Future studies, including those involving different genders and ethnicities, are needed in order to obtain more detailed insights into the biological resilience of SSCs.

Acknowledgments We would like to thank all the members of SONIC for their thoughtful advice and helpful discussions, and Dr. Yoshikawa (Infocom Co.) for technical assistance with O-PLS. The members of SONIC are: Dr. Yasumichi Arai (Keio University School of Medicine), Dr. Yasuyuki Gondo (Osaka University Graduate School of Human Sciences), Dr. Kazunori Ikebe (Osaka University Graduate School of Dentistry), Dr. Tatsuro Ishizaki (Tokyo Metropolitan Institute of Gerontology), Dr. Kei Kamide (Osaka University School of Medicine), Dr. Yukie Masui (Tokyo Metropolitan Institute of Gerontology), and Dr. Ryutaro Takahashi (Tokyo Metropolitan Institute of Gerontology).

Author Contributions Conceived and designed the experiments: TE YM N. Hashii. Performed the experiments: YM N. Hashii DT YO Y. Abe. Analyzed the data: YM N. Hashii HT Y. Arai. Contributed reagents/materials/analysis tools: Y. Abe Y. Arai N. Hirose. Wrote the paper: TE YM N. Hashii Y. Arai NK N. Hirose.