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A primary pathology of Alzheimer’s disease (AD) is Aβ deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aβ precursor protein (APP), but vascular amyloid deposits’ origins remain unclear. Endothelial APP expression in APP-knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP’s importance. Furthermore, two types of endothelial APP—with and without O-glycans—have been biochemically identified, but only the former is cleaved for Aβ production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that APP lacking O-glycans is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Αβ production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00851-1.

Glycosylation of the conserved asparagine residue in each heavy chain of IgG in the CH2 domain is known as N-glycosylation. It is one of the most common post-translational modifications and important critical quality attributes of monoclonal antibody (mAb) therapeutics. Various studies have demonstrated the effects of the Fc N-glycosylation on safety, Fc effector functions, and pharmacokinetics, both dependent and independent of neonatal Fc receptor (FcRn) pathway. However, separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and existing studies often discuss the overall impact of N-glycans, without considering the individual contributions of each glycoform when evaluating mAbs with highly heterogeneous distributions. In this study, chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity was used to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. We demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibody-dependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.

We developed two human-induced pluripotent stem cell (hiPSC)/human embryonic stem cell (hESC)-specific glycan-recognizing mouse antibodies, R-10G and R-17F, using the Tic (JCRB1331) hiPSC line as an antigen. R-10G recognizes a low-sulfate keratan sulfate, and R-17F recognizes lacto-N-fucopentaose-1. To evaluate the general characteristics of stem cell glycans, we investigated the hiPSC line 201B7 (HPS0063), a prototype iPSC line. Using an R-10G affinity column, an R-10G-binding protein was isolated from 201B7 cells. The protein yielded a single but very broad band from 480 to 1236 kDa by blue native gel electrophoresis. After trypsin digestion, the protein was identified as podocalyxin by liquid chromatography/mass spectrometry. According to Western blotting, the protein reacted with R-10G and R-17F. The R-10G-positive band was resistant to digestion with glycan-degrading enzymes, including peptide N-glycanase, but the intensity of the band was decreased significantly by digestion with keratanase, keratanase II, and endo-β-galactosidase, suggesting the R-10G epitope to be a keratan sulfate. These results suggest that keratan sulfate-type epitopes are shared by hiPSCs. However, the keratan sulfate from 201B7 cells contained a polylactosamine disaccharide unit (Galβ1-4GlcNAc) at a significant frequency, whereas that from Tic cells consisted mostly of keratan sulfate disaccharide units (Galβ1-4GlcNAc(6S)). In addition, the abundance of the R-10G epitope was significantly lower in 201B7 cells than in Tic cells.

Fc-receptors for immunoglobulin G (FcγRs) mediate a variety of effector and regulatory mechanisms in the immune system. N-glycosylation of FcγRs critically affects their functions which is well exemplified by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis mediated by homologous FcγRIIIa and FcγRIIIb, respectively. Although several reports describe N-glycosylation profiles of recombinant FcγRIII glycoproteins, much remains unknown regarding their native glycoforms. Here we performed site-specific N-glycosylation profiling of a soluble form of FcγRIIIb purified from human serum based on mass spectrometric analysis. Our data indicate a distinct and common tendency of the glycoforms exhibited at each N-glycosylation site between the native and the previously reported recombinant FcγRIII glycoproteins. Among the six N-glycosylation sites of serum soluble FcγRIIIb, Asn45 was shown to be exclusively occupied by high-mannose-type oligosaccharides, whereas the remaining sites were solely modified by the complex-type oligosaccharides with sialic acid and fucose residues. The results of our endogenous FcγRIII glycoform analyses are important for the optimization of therapeutic antibody efficacy.

In the mammalian nervous system, protein N-glycosylation plays an important role in neuronal physiology. In this study, we performed a comprehensive N-glycosylation analysis of mouse GluA1, one of the major subunits of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate type glutamate receptor, which possesses six potential N-glycosylation sites in the N-terminal domain. By mass spectrometry-based analysis, we identified the N-glycoforms and semiquantitatively determined the site-specific N-glycosylation occupancy of GluA1. In addition, only the N401-glycosylation site demonstrated incomplete N-glycosylation occupancy. Therefore, we generated a peptide antibody that specifically detects the N401-glycan-free form to precisely quantify N401-glycosylation occupancy. Using this antibody, we clarified that N401 occupancy varies between cell types and increases in an age-dependent manner in mouse forebrains. To address the regulatory mechanism of N401-glycosylation, binding proteins of GluA1 around the N401 site were screened. HSP70 family proteins, including Bip, were identified as candidates. Bip has been known as a molecular chaperone that plays a key role in protein folding in the ER (endoplasmic reticulum). To examine the involvement of Bip in N401-glycosylation, the effect of Bip over-expression on N401 occupancy was evaluated in HEK293T cells, and the results demonstrated Bip increases the N401 glycan-free form by mediating selective prolongation of its protein half-life. Taken together, we propose that the N401-glycosite of GluA1 receives a unique control of modification, and we also propose a novel N-glycosylation occupancy regulatory mechanism by Bip that might be associated with α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors function in the brain.

Recently, we established two mouse monoclonal antibodies (R-10G and R-17F). The R-17F antibody (IgG1 subtype) exhibited a strong cytotoxic effect on hiPS/ES cells. The R-17F antigen isolated from a total lipid extract of hiPS (Tic) cells was identified as LNFP I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc). In the present study, R-17F binding proteins were isolated from hiPS (Tic) cell lysates with an affinity column of R-17F. They gave one major R-17F positive band around 250 kDa, and several minor bands between 150 kDa and 25 kDa. The former band was identified as podocalyxin by LC/MS/MS after SDS-PAGE. Hapten inhibition studies on R-17F binding to R-17F column-purified proteins with various synthetic oligosaccharides revealed that the blood group H type 1 triaose structure (Fucα1-2Galβ1-3GlcNAc) was the predominant epitope on all the R-17F binding proteins. These bands disappeared completely on digestion with α1-2 fucosidase, but not with α1-3/4 fucosidase. Upon PNGase F digestion, the R-17F positive band around and above 250 kDa did not show any change, while the minor bands between 150 kDa and 25 kDa disappeared completely, suggesting that the epitope is expressed on N-glycans in the latter and probably on O-glycans in the former. These results, together with those obtained in our previous studies on R-10G (Kawabe et al. Glycobiology, 23, 322-336 (2013)), indicated that both R-10G and R-17F epitopes are carried on the same podocalyxin molecule. The R-17F epitopes on these glycoproteins expressed on hiPS cells could be associated with the molecular mechanism underlying the carbohydrate-mediated cytotoxic activity of R-17F.

Some aberrant N-glycosylations are being used as tumor markers, and glycoproteomics is expected to provide novel diagnosis markers and targets of drug developments. However, one has trouble in mass spectrometric glycoproteomics of membrane fraction because of lower intensity of glycopeptides in the existence of surfactants. Previously, we developed a glycopeptide enrichment method by acetone precipitation, and it was successfully applied to human serum glycoproteomics. In this study, we confirmed that this method is useful to remove the surfactants and applicable to membrane glycoproteomics. The glycoproteomic approach to the human fetal lung fibroblasts membrane fraction resulted in the identification of over 272 glycoforms on 63 sites of the 44 glycoproteins. According to the existing databases, the structural features on 41 sites are previously unreported. The most frequently occurring forms at N-glycosylation site were high-mannose type containing nine mannose residues (M9) and monosialo-fucosylated biantennary oligosaccharides. Several unexpected N-glycans, such as fucosylated complex-type and fucosylated high-mannose and/or fucosylated pauci-mannose types were found in ER and lysosome proteins. Our method provides new insights into transport, biosynthesis, and degradation of glycoproteins.

During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders.

A class of glycoproteins such as carcinoembryonic antigen (CEA)/CEA-related cell adhesion molecule 1(CEACAM1), CD26 (DPPIV), and mac-2 binding protein (Mac-2BP) harbor tumor-associated glycans in colorectal cancer. In this study, we identified type II transmembrane mosaic serine protease large-form (MSPL) and its splice variant transmembrane protease serine 13 (TMPRSS13) as ligands of Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on the colorectal cancer cells. DC-SIGN is a C-type lectin expressed on dendritic cells, serves as a pattern recognition receptor for numerous pathogens such as human immunodeficiency virus (HIV) and M. tuberculosis. DC-SIGN recognizes these glycoproteins in a Ca2+ dependent manner. Meanwhile, we found that MSPL proteolytically cleaves DC-SIGN in addition to the above glycan-mediated recognition. DC-SIGN was degraded more efficiently by MSPL when treated with ethylenediaminetetraacetic acid (EDTA), suggesting that glycan-dependent interaction of the two molecules partially blocked DC-SIGN degradation. Our findings uncovered a dual recognition system between DC-SIGN and MSPL/TMPRSS13, providing new insight into the mechanism underlying colorectal tumor microenvironment.

Background Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. Methods Plasma proteins from Japanese semi-supercentenarians (SSCs, 106–109 years), aged controls (70–88 years), and young controls (20–38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. Results We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. Conclusions Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. General significance We found abundant glycans in SSCs, which may be associated with human longevity.

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

Therapeutic monoclonal antibodies (mAbs) exert their effects via binding to specific target molecules, which is expected to show rare off-target adverse reactions. However, nonclinical evaluation of mAbs is difficult because they often lack reactivity toward orthologous targets in animals. During the nonclinical evaluation of mAbs, not only the target molecules but fragment crystallizable (Fc) receptors, which regulate the immune effector functions and pharmacokinetic properties of mAbs, should be considered. In this review, factors for extrapolating nonclinical study results to clinical settings are discussed by focusing on Fc receptors. The human Fcγ receptor family consists of FcγRI, IIa, IIb, IIIa, and IIIb; Fcγ receptors in laboratory animals are structurally and functionally different from those in humans. In addition, interactions between human IgG-Fc, a component of therapeutic mAbs, and animal Fcγ receptors are still not fully understood. With regard to neonatal Fc receptor (FcRn), related molecules comprising the FcRn family are not known; however, critical amino acid residues involved in IgG binding are different between human and mouse. In case of next-generation mAbs with a novel structure or mode of action, knowledge from related drugs is limited. To ensure safety of next-generation mAbs, a thorough understanding of the differences in Fc receptors among species and the interactions between mAbs and Fc receptors is required, and the appropriateness of the nonclinical study design should be carefully examined prior to conducting clinical studies.

Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing (15)N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a (15)N-enrichment ratio of approximately 80%. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.

Many monoclonal antibodies have been developed for therapy over the last 2 decades. In the development of therapeutic antibodies, the preclinical assessment of an antibody's biodistribution is important for the prediction of the antibody's efficacy and safety. For imaging analyses of such biodistributions, radioisotope (RI) labeling and fluorescence labeling methods are typically used, but the resulting data are limited because these methods cannot distinguish breakdown products from intact antibodies. To resolve this problem, we developed a novel method using fluorescent resonance energy transfer (FRET)-type labeling and a spectral unmixing tool. With FRET-type labeling (labeling with 2 species of fluorophore), different fluorescence properties of labeled intact antibodies and their breakdown products (the hydrolyzed/digested type of breakdown products) are made visible. With the spectral unmixing tool, the fluorescence of a solution containing the intact antibody and its breakdown products could be unmixed in proportion to their contents. Moreover, when labeled antibodies that targeted either human epidermal growth factor receptor-2 or epidermal growth factor receptor were injected into nude mice implanted subcutaneously with tumor cells, the accumulation of the injected labeled antibodies and their breakdown products in the tumor could be separately analyzed by both whole-mouse imaging and a tumor homogenate analysis. These results suggest that our method using FRET-type labeling and a spectral unmixing tool could be useful in distinguishing breakdown products from intact antibodies.

This study was undertaken to evaluate the performance of anti-drug antibody (ADA) assays constructed by each participating company using common samples including ADA, drug and human serum. The ADA assays constructed by each company showed good sensitivity and precision for evaluation of ADA. Cut points for screening and confirmatory assays and assay selectivity were determined by various calculation methods. In evaluations of blind ADA samples, nearly similar results were obtained by the study companies in determinations of whether samples were positive or negative except at the lowest sample concentration (5 ng/mL). In measurement of drug tolerance, for almost samples containing ADA and drugs, more positive results were obtained in assays using acid dissociation compared to those without acid dissociation. Overall, the performance of ADA assays constructed by the 10 companies participating in this study was acceptable in terms of sensitivity and reproducibility for detection and evaluation of immunogenicity in both patients and healthy subjects. On the other hand, based on results for samples containing ADA and drugs, validity of results for ADA assays conducted without acid dissociation was less meaningful and more difficult to evaluate. Thus, acid dissociation was confirmed to be useful for improving drug tolerance.

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder., Author summary Ngly1 is a cytoplasmic de-N-glycosylating enzyme that is ubiquitously found in eukaryotes. This enzyme is involved in a process referred to as endoplasmic reticulum-associated degradation (ERAD), one of the quality control mechanisms for newly synthesized proteins. A genetic disorder, NGLY1-deficiency, caused by mutations in the NGLY1 gene has recently been discovered. However, the precise mechanism for the pathogenesis of this devastating disease continues to remain unclear. We report herein that Ngly1-deficient mice are embryonically lethal in a C57BL/6 background. Surprisingly, the lethality was suppressed by crossing the mice with an outbred mouse strain (ICR), suggesting that the phenotypic consequence of Ngly1 is greatly influenced by their genetic background. In both cases, the additional deletion of Engase in Ngly1-deficient mice could strongly mitigate the phenotypes. Interestingly, the remaining defects in Ngly1-deficient or Ngly1/Engase-deficient mice were reminiscent of the symptoms of subjects with an NGLY1-deficiency. Our results clearly point to the importance of Ngly1 in mammals and show that the inhibition of ENGase represents an effective therapy for treating an NGLY1-deficiency. Most importantly, the mice described herein could serve as valuable viable model mice for studies related to the pathophysiology of an NGLY1-deficiency.

Because the ionization efficiency for glycopeptides is lower than that of peptides in electrospray ionization, it is frequently necessary to enrich them prior to their analysis using liquid chromatography coupled with tandem mass spectrometry. Although some methods for selectively enriching glycopeptides (e.g., lectin, agarose, and cellulose methods) have been reported, they are time-consuming (procedures that require several hours) and may not be applicable to submicrogram-sized samples. Here, we report on a rapid, simple method for enriching glycopeptides in small sample amounts using cellulose hydrophilic interaction (cellulose HILIC)/reversed-phase (RP) stop-and-go extraction tips (StageTips). Using the cellulose HILIC/RP StageTips, glycopeptide-selective enrichment can be achieved at the nanogram level within a few minutes.

RATIONALE Glycan heterogeneity on recombinant human erythropoietin (rEPO) product is considered to be one of the critical quality attributes, and similarity tests of glycan heterogeneities are required in the manufacturing process changes and developments of biosimilars. A method for differentiating highly complex and diverse glycosylations is needed to evaluate comparability and biosimilarity among rEPO batches and products manufactured by different processes. METHODS The glycan heterogeneities of nine rEPO products (four innovator products and five biosimilar products) were distinguished by multivariate analysis (MVA) using the peak area ratios of each glycan to the total peak area of glycans in mass spectra obtained by liquid chromatography/mass spectrometry (LC/MS) of N-glycans from rEPOs. RESULTS Principal component analysis (PCA) using glycan profiles obtained by LC/MS proved to be a useful method for differentiating glycan heterogeneities among nine rEPOs. Using PC values as indices, we were able to visualize and digitalize the glycan heterogeneities of each rEPO. The characteristic glycans of each rEPO were also successfully identified by orthogonal partial least-squares discrimination analysis (OPLS-DA), an MVA method, using the glycan profile data. CONCLUSIONS PCA values were useful for evaluating the relative differences among the glycan heterogeneities of rEPOs. The characteristic glycans that contributed to the differentiation were also successfully identified by OPLS-DA. PCA and OPLS-DA based on mass spectrometric data are applicable for distinguishing glycan heterogeneities, which are virtually indistinguishable on rEPO products. Copyright © 2014 John Wiley & Sons, Ltd.

The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose β1, 2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.

The recent patent expirations of erythropoietin (EPO) have promoted the development of biosimilars. Two and one biosimilar EPO products were approved in 2007 in Europe and in 2010 in Japan, respectively. Glycosylation heterogeneity of EPO is very complex, and its pattern has a large impact on its in vivo activity. In this study, glycoform profilings of biosimilar and innovator EPO products were performed using LC/ESI-MS. Glycoforms of EPO were detected within the range of m/z 1700-3600 at the 10(+)-16(+) charge states. The charge-deconvoluted spectra showed complex glycoform mass profiles at 28,000-32,000 Da, and most of the observed peaks were assigned to the peptide (18,236 Da)+glycans with the compositions of NeuAc10-14Hexn+3HexNAcnFuc3 (n=16-26) with or without some O-acetylations (+42 Da) and attachment of NeuGc for NeuAc or oxidation (+16 Da). Analysis of de-N-glycosylated EPO showed the distributions of O-glycans of NeuAc1-2Hex1HexNAc1 and site occupancy. Each EPO product showed a characteristic glycoform profile with respect to sialylation, glycan size, O-acetylation of sialic acids and O-glycosylation. Analysis of darbepoetin suggested that glycans of darbepoetin were highly sialylated and O-acetylated. LC/ESI-MS was shown to be useful to evaluate the similarity of the glycoform profiles of EPO.

Human insulin and insulin lispro (lispro), a rapid-acting insulin analog, have identical primary structures, except for the transposition of a pair of amino acids. This mutation results in alterations in their higher order structures, with lispro dissociating more easily than human insulin. In our previous study performed using hydrogen/deuterium exchange mass spectrometry (HDX/MS), differences were observed in the rates and levels of deuteration among insulin analog products, which were found to be related to their self-association stability. In this study, we carried out peptide mapping of deuterated human insulin and lispro to determine the regions responsible for these deuteration differences and to elucidate the type of structural changes that affect their HDX reactivity. We identified A3-6 and B22-24 as the 2 regions that showed distinct differences in the number of deuterium atoms incorporated between human insulin and lispro. These regions contain residues that are thought to participate in hexamerization and dimerization, respectively. We also determined that over time, the differences in deuteration levels decreased in A3-6, whereas they increased in B22-24, suggesting a difference in the dynamics between these 2 regions. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

Background CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography. Methods In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10. Results CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn628-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. Conclusions N-glycosylation at Asn628 is essential not only for NEP activities, but also for surface expression. General significance Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane.

Rheumatoid arthritis (RA)-associated IgG antibodies such as anti-citrullinated protein antibodies (ACPAs) have diverse glycosylation variants; however, key sugar chains modulating the arthritogenic activity of IgG remain to be clarified. Here, we show that reduced sialylation is a common feature of RA-associated IgG in humans and in mouse models of arthritis. Genetically blocking sialylation in activated B cells results in exacerbation of joint inflammation in a collagen-induced arthritis (CIA) model. On the other hand, artificial sialylation of anti-type II collagen antibodies, including ACPAs, not only attenuates arthritogenic activity, but also suppresses the development of CIA in the antibody-infused mice, whereas sialylation of other IgG does not prevent CIA. Thus, our data demonstrate that sialylation levels control the arthritogenicity of RA-associated IgG, presenting a potential target for antigen-specific immunotherapy., Post-translational modifications, such as glycosylation and sialylation, are thought to confer disease modifying effects on autoimmune-associated antibodies, including anti-citrullinated protein antibodies in rheumatoid arthritis. Here the authors show that sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis in mice.