IgLON cell adhesion molecules regulate synaptogenesis in hippocampal neurons

IgLON cell adhesion molecules (CAMs) belonging to the immunoglobulin superfamily comprise of LAMP, neurotrimin (Ntm), OBCAM, and Kilon. In the present study, we performed the single and double transfection of IgLON gene constructs into hippocampal neurons in vitro and evaluated synaptic number. The quantitative analysis showed that the single over‐expression of LAMP or OBCAM increased synaptic number, while the over‐expression of Kilon reduced synaptic number and Ntm had no effects. The double over‐expression of Kilon‐Ntm, Kilon‐OBCAM, LAMP‐Ntm, and Ntm‐OBCAM decreased synaptic number and that of Kilon‐LAMP and LAMP‐OBCAM had no effect. These results suggest that IgLON CAMs participate in regulating synapse formation in hippocampal neurons. Copyright © 2009 John Wiley & Sons, Ltd.     

Overexpression of IgLON cell adhesion molecules changes proliferation and cell size of cortical astrocytes

IgLON family is a subgroup of the immunoglobulin superfamily cell adhesion molecules and composed of limbic system-associated protein (LAMP), opioid binding cell adhesion molecule (OBCAM), neurotrimin (Ntm) and Kilon. In the present study, we investigated the overexpression of LAMP, OBCAM, Ntm and Kilon on the proliferation and cell size of type-1 astrocytes in vitro. Quantitative analysis using bromodeoxyuridine immunocytochemistry revealed that the expression of OBCAM had greater inhibitory effect on astrocytic proliferation as compared with LAMP, Ntm and Kilon ones. OBCAM overexpression increased the cell size of astrocytes as compared with the control. The treatment of FGF-2 had greater proliferative effect on OBCAM-transfected astrocytes as compared with the control. These results suggest that OBCAM is more potent regulator for controlling the proliferation and cell size of astrocytes as compared with other IgLON proteins possibly through FGF-2 receptor-mediated pathway.     

Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family

In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called "raft." In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a kindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.     

Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP

Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic "barreloids." Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively.     

cDNA cloning of the CEPUS, a secreted type of neural glycoprotein belonging to the immunoglobulin-like opioid binding cell adhesion molecule (OBCAM) subfamily.

GP55 is a family of glycoproteins distributed predominantly in the nervous system, and its previously characterized members, including the GP55A (EMBL Y08170) and E19S (EMBL Y08171) reveal a typical glycosyl phosphatidyl inositol (GPI)-anchored pattern for membrane proteins. CEPUS identified in this study appeared to represent the third member of GP55. This 3.2 kb long complete cDNA clone from the chicken brain exhibited 3 Ig-like domains. The open reading frame of CEPUS contains 313 amino acids, which can encode a 31.7 kDa core protein (pI 5.75) for the mature form. The signal peptide cleavage site was predicted at Gln25. The structural features of the CEPUS cDNA sequence represented a soluble counterpart to the recently identified cerebellar Purkinje cell specific antigen, CEPU-1. The sequence difference between CEPU-1 and CEPUS was only found in the C-terminus in which the CEPUS lacked the GPI-anchored binding site. It displays significant sequence homology to GP55-related molecules, including OBCAM, GP55A, E19S/LAMP, neurotrimin, and CEPU-1, which are all membrane attached types. The absence of the hydrophobic tail sequence in CEPUS may, therefore, suggest that CEPUS would represent the first identified secreted member in this group of genes. We defined that this molecule forms the opioid-binding cell adhesion molecule (OBCAM) subfamily in the molecular phylogeny. Structurally, these molecules represent acidic proteins (pI 5.47-6.09). Six cysteins, as well as 5 Asn-linked potential glycosylation sites were evolutionary-conserved, suggesting that this OBCAM subfamily resembles immunoglobulin-like and highly glycosylated molecules. The presence of CEPUS would probably suggest to us that the spatial/local expression of the CEPU gene may provide a favorable route for migrating CEPU-positive population of neurons to generate a neuron-specific guidance in developing neurons in vivo.     

Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.     

Promotion of neuronal cell adhesion by members of the IgLON family occurs in the absence of either support or modification of neurite outgrowth

The IgLONs are a family of glycosyl phosphatidyl inositol-linked cell adhesion molecules which are thought to modify neurite outgrowth and may play a role in cell-cell recognition. The family consists of LAMP, OBCAM, neurotrimin/CEPU-1 and neurotractin/kilon. In this paper we report the effect of recombinant LAMP, CEPU-1 and OBCAM, and transfected cell lines expressing these molecules, on the adhesion and outgrowth of dorsal root ganglion (DRG) and sympathetic neurones. CHO cells transfected with cDNA for CEPU-1 adhered to a recombinant CEPU-1-Fc substrate. However, DRG or sympathetic neurones only adhered to CEPU-1-Fc when presented on protein A. Although DRG and sympathetic neurones express IgLONs on their surface, both types of neurones exhibited differential adhesion to CEPU-1-Fc, LAMP-Fc and OBCAM-Fc. Neither DRG nor sympathetic neurones extended neurites on a protein A/IgLON-Fc substrate and overexpression of CEPU-1-GFP in DRG neurones also failed to stimulate neurite outgrowth on an IgLON-Fc substrate. DRG neurones adhered to and extended neurites equally on transfected and non-transfected cell lines and the recombinant proteins did not modulate the outgrowth of neurones on laminin. In contrast to previous reports we suggest that IgLONs may not have a primary role in axon guidance but may be more important for cell-cell adhesion and recognition.     

IgLON Cell Adhesion Molecules Are Shed from the Cell Surface of Cortical Neurons to Promote Neuronal Growth

Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons.     

Dolichol-phosphate mannose synthase: structure, function and regulation

Glycosylation is the major modification of proteins, and alters their structures, functions and localizations. Glycosylation of secretory and surface proteins takes place in the endoplasmic reticulum and Golgi apparatus in eukaryotic cells and is classified into four modification pathways, namely N- and O-linked glycosylations, glycosylphosphatidylinositol (GPI)-anchor and C-mannosylation. These modifications are accomplished by sequential addition of single monosaccharides (O-linked glycosylation and C-mannosylation) or en bloc transfer of lipid-linked oligosaccharides (N-linked glycosylation and GPI) onto the proteins. The glycosyltransferases involved in these glycosylations are categorized into two classes based on the type of sugar donor, namely nucleotide-sugars and dolichol-phosphate-sugars, in which the sugar moiety is mannose or glucose. The sugar transfer from dolichol-phosphate-sugars occurs exclusively on the luminal side of the endoplasmic reticulum and is utilized in all four glycosylation pathways. In this review, we focus on the biosynthesis of dolichol-phosphate-mannose, and particularly on the mammalian enzyme complex involved in the reaction.     

Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography-mass spectrometry.

A mass spectrometry method has been developed for selective detection of glycopeptides at the low (< or = 25) picomole level during chromatography of glycoprotein digests and for differentiation of O-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium-ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate-related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in Q2, the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc+). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) "parent-scan" spectra. N-linked carbohydrates are differentiated from O-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase F. These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42-kDa glycoprotein containing three N-linked, and at least three O-linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC-ESMS studies.     

Glycopeptide profiling of beta-2-glycoprotein I by mass spectrometry reveals attenuated sialylation in patients with antiphospholipid syndrome

β2-glycoprotein I (β2GPI) is a five-domain protein associated with the antiphospholipid syndrome (APS), however, its normal biological function is yet to be defined. β2GPI is N-glycosylated at several asparagine residues and the glycan moiety conjugated to residue 143 has been proposed to interact with the Gly40–Arg43 motif of β2GPI. The Gly40–Arg43 motif has also been proposed to serve as the epitope for the anti-β2GPI autoantibody associated with APS. We hypothesized that the structure or composition of the glycan at Asn-143 might be associated with the APS symptom by shielding or exposing the Gly40–Arg43 motif towards the anti-β2GPI autoantibody. To test this hypothesis we used mass spectrometry (MS) for comparative glycopeptide profiling of human β2GPI obtained from blood serum from four healthy test subjects and six APS patients. It revealed significant differences in the extent of sialylation and branching of glycans at Asn-143. Biantennary glycans were more abundant than triantennary glycans at Asn-143 in both healthy subjects and patients. In APS patient samples we observed a decrease in sialylated triantennary glycans and an increase in sialylated biantennary glycan structures, as compared to controls. These data indicate that some APS patients have β2GPI molecules with a reduced number of negatively charged sialic acid units in the glycan structure at Asn-143. This alteration of the electrostatic properties of the glycan moiety may attenuate the intramolecular interactions with the positively charged Gly40–Arg43 motif of β2GPI and, in turn, leads to conformational instability and exposure of the disease-related linear epitope Gly40–Arg43 to the circulating autoantibody. Thus, our study suggests a link between site-specific glycan profiles of β2GPI and the pathology of antiphospholipid syndrome.     

Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.     

Glycosylphosphatidylinositol biosynthesis defects in Gpi11p- and Gpi13p-deficient yeast suggest a branched pathway and implicate gpi13p in phosphoethanolamine transfer to the third mannose

Glycosylphosphatidylinositols (GPIs) are critical for membrane anchoring and intracellular transport of certain secretory proteins. GPIs have a conserved trimannosyl core bearing a phosphoethanolamine (EthN-P) moiety on the third mannose (Man-3) through which the glycolipid is linked to protein, but diverse GPI precursors with EthN-Ps on Man-1 and Man-2 have also been described. We report on two essential yeast genes whose products are required late in GPI assembly. GPI11 (YDR302w) encodes a homologue of human Pig-Fp, a protein implicated in the addition of EthN-P to Man-3. PIG-F complements the gpi11 deletion, but the rescued haploids are temperature sensitive. Abolition of Gpi11p or Pig-Fp function in GPI11 disruptants blocks GPI anchoring and formation of complete GPI precursors and leads to accumulation of two GPIs whose glycan head groups contain four mannoses but differ in the positioning and number of side chains, probably EthN-Ps. The less polar GPI bears EthN-P on Man-2, whereas the more polar lipid has EthN-P on Man-3. The latter finding indicates that Gpi11p is not required for adding EthN-P to Man-3. Gpi13p (YLL031cp), a member of a family of phosphoryltransferases, is a candidate for the enzyme responsible for adding EthN-P to Man-3. Depletion of Gpi13p in a Gpi11p-defective strain prevents formation of the GPI bearing EthN-P on Man-3, and Gpi13p-deficient strains accumulate a Man(4)-GPI isoform that bears EthN-P on Man-1. We further show that the lipid accumulation phenotype of Gpi11p-deficient cells resembles that of cells lacking Gpi7p, a sequence homologue of Gpi13p known to add EthN-P to Man-2 of a late-stage GPI precursor. This result suggests that in yeast a Gpi11p-deficiency can affect EthN-P addition to Man-2 by Gpi7p, in contrast to the Pig-Fp defect in mammalian cells, which prevents EthN-P addition to Man-3. Because Gpi11p and Pig-Fp affect EthN-P transfer to Man-2 and Man-3, respectively, these proteins may act in partnership with the GPI-EthN-P transferases, although their involvement in a given EthN-P transfer reaction varies between species. Possible roles for Gpi11p in the supply of the EthN-P donor are discussed. Because Gpi11p- and Gpi13p-deficient cells accumulate isoforms of Man(4)-GPIs with EthN-P on Man-2 and on Man-1, respectively, and because the GPIs that accumulate in Gpi11p-defective strains are likely to have been generated independently of one another, we propose that the yeast GPI assembly pathway is branched.     

Proteomics analysis of serum from mutant mice reveals lysosomal proteins selectively transported by each of the two mannose 6-phosphate receptors

Most mammalian cells contain two types of mannose 6-phosphate (Man-6-P) receptors (MPRs): the 300 kDa cation-independent (CI) MPR and 46 kDa cation-dependent (CD) MPR. The two MPRs have overlapping function in intracellular targeting of newly synthesized lysosomal proteins, but both are required for efficient targeting. Despite extensive investigation, the relative roles and specialized functions of each MPR in targeting of specific proteins remain questions of fundamental interest. One possibility is that most Man-6-P glycoproteins are transported by both MPRs, but there may be subsets that are preferentially transported by each. To investigate this, we have conducted a proteomics analysis of serum from mice lacking either MPR with the reasoning that lysosomal proteins that are selectively transported by a given MPR should be preferentially secreted into the bloodstream in its absence. We purified and identified Man-6-P glycoproteins and glycopeptides from wild-type, CDMPR-deficient, and CIMPR-deficient mouse serum and found both lysosomal proteins and proteins not currently thought to have lysosomal function. Different mass spectrometric approaches (spectral count analysis of nanospray LC-MS/MS experiments on unlabeled samples and LC-MALDI/TOF/TOF experiments on iTRAQ-labeled samples) revealed a number of proteins that appear specifically elevated in serum from each MPR-deficient mouse. Man-6-P glycoforms of cellular repressor of E1A-stimulated genes 1, tripeptidyl peptidase I, and heparanase were elevated in absence of the CDMPR and Man-6-P glycoforms of alpha-mannosidase B1, cathepsin D, and prosaposin were elevated in the absence of the CIMPR. Results were confirmed by Western blot analyses for select proteins. This study provides a comparison of different quantitative mass spectrometric approaches and provides the first report of proteins whose cellular targeting appears to be MPR-selective under physiological conditions.     

Constraints on the transport and glycosylation of recombinant IFN-gamma in Chinese hamster ovary and insect cells.

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.     

Tools for glycomics: relative quantitation of glycans by isotopic permethylation using 13CH3I

Analysis of oligosaccharides by mass spectrometry (MS) has enabled the investigation of the glycan repertoire of organisms with high resolution and sensitivity. It is difficult, however, to correlate the expression of glycosyltransferases with the glycan structures present in a particular cell type or tissue because the use of MS for quantitative purposes has significant limitations. For this reason, in order to develop a technique that would allow relative glycan quantification by MS analysis between two samples, a procedure was developed for the isotopic labeling of oligosaccharides with (13)C-labeled methyl iodide using standard permethylation conditions. Separate aliquots of oligosaccharides from human milk were labeled with (12)C or (13)C methyl iodide; the labeled and non-labeled glycans were mixed in known proportions, and the mixtures analyzed by MS. Results indicated that the isotopic labeling described here was capable of providing relative quantitative data with a dynamic range of at least two orders of magnitude, adequate linearity, and reproducibility with a coefficient of variation that was 13% on average. This procedure was used to analyze N-linked glycans released from various mixtures of glycoproteins, such as alpha-1 acid glycoprotein, human transferrin, and bovine fetuin, using MS techniques that included matrix assisted laser desorption ionization-time of flight MS and electrospray ionization with ion cyclotron resonance-Fourier transformation MS. The measured (12)C:(13)C ratios from mixtures of glycans permethylated with either (12)CH(3)I or (13)CH(3)I were consistent with the theoretical proportions. This technique is an effective procedure for relative quantitative glycan analysis by MS.     

Site-specific glycosylation analysis of human apolipoprotein B100 using LC/ESI MS/MS

Human apolipoprotein B100 (apoB100) has 19 potential N-glycosylation sites, and 16 asparagine residues were reported to be occupied by high-mannose type, hybrid type, and monoantennary and biantennary complex type oligosaccharides. In the present study, a site-specific glycosylation analysis of apoB100 was carried out using reversed-phase high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI MS/MS). ApoB100 was reduced, carboxymethylated, and then digested by trypsin or chymotrypsin. The complex mixture of peptides and glycopeptides was subjected to LC/ESI MS/MS, where product ion spectra of the molecular ions were acquired data-dependently. The glycopeptide ions were extracted and confirmed by the presence of carbohydrate-specific fragment ions, such as m/z 204 (HexNAc) and 366 (HexHexNAc), in the product ion spectra. The peptide moiety of glycopeptide was determined by the presence of the b- and y-series ions derived from its amino acid sequence in the product ion spectrum, and the oligosaccharide moiety was deduced from the calculated molecular mass of the oligosaccharide. The heterogeneity of carbohydrate structures at 17 glycosylation sites was determined using this methodology. Our data showed that Asn2212, not previously identified as a site of glycosylation, could be glycosylated. It was also revealed that Asn158, 1341, 1350, 3309, and 3331 were occupied by high-mannose type oligosaccharides, and Asn 956, 1496, 2212, 2752, 2955, 3074, 3197, 3438, 3868, 4210, and 4404 were predominantly occupied by mono- or disialylated oligosaccharides. Asn3384, the nearest N-glycosylation site to the LDL-receptor binding site (amino acids 3359-3369), was occupied by a variety of oligosaccharides, including high-mannose, hybrid, and complex types. These results are useful for understanding the structure of LDL particles and oligosaccharide function in LDL-receptor ligand binding.     

A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M]⁺ ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K]⁺ and [M + Na]⁺ were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.     

Application of liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin

High-performance liquid chromatography with electrospray ionization mass spectrometry (LC/MS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS) were applied to the analysis of the site-specific carbohydrate heterogeneity in erythropoietin (EPO) used as a model of the sialylated glycoprotein. N-linked oligosaccharides were released from recombinant human EPO expressed in Chinese hamster ovary cells enzymatically and reduced with NaBH(4). Many different sialylated oligosaccharides of EPO were separated and characterized by LC/MS equipped with a graphitized carbon column (GCC). Glycosylation sites and the preliminary glycosylation pattern at each glycosylation site were determined by LC/MS of endoproteinase Glu-C-digested EPO. The detailed site-specific carbohydrate heterogeneity caused by the differences in the molecular weight, branch, linkage, and sequence was elucidated by GCC-LC/MS of the N-linked oligosaccharides released from the isolated glycopeptides. Structural details of the isomers were analyzed by LC/MS/MS, and it was indicated that di- and trisialylated tetraantennary oligosaccharides are attached to Asn24, 38, and 83, whereas their isomers, di- and trisialylated triantennary oligosaccharides containing N-acetyllactosamines, are combined with Asn24. Our method is useful for the determination of glycosylation sites, the site-specific carbohydrate heterogeneity of glycoproteins, and the carbohydrate structure.