Plant cultured cells expressing human beta1,4-galactosyltransferase secrete glycoproteins with galactose-extended N-linked glycans

Previously, we generated transgenic tobacco BY2 suspension-cultured cells (GT6 cells) that produced human beta1,4-galactosyltransferase. In this study, we analyze the N-glycan structures of glycoproteins secreted from GT6 cells to the spent medium. The N-glycans were liberated by hydrazinolysis, and the resulting oligosaccharides were labeled with 2-aminopyridine (PA). The pyridylaminated glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by the combined use of 2D PA sugar chain mapping, MS/MS analysis, and exoglycosidase digestion. The distribution of proposed N-glycan structures of GT6-secreted glycoproteins (GalGNM5 [26.8%], GalGNM4 [18.4%], GalGNM3 [19.6%], and GalGNM3X [35.2%]) is different from that found in intracellular glycoproteins (M7A [9.3%], M7B [15.9%], M6B [19.5%], M5 [1.4%], M3X [6.6%], GalGNM5 [35.5%], and GalGNM3 [11.8%]). In vitro, sialic acid was transferred to sugar chains of extracellular glycoproteins from the GT6 spent medium. The results suggest that sugar chains of extracellular glycoproteins from the GT6 spent medium are candidates for substrates of sialic acid transfer.     

Natural antibodies to glycans

A wide variety of so-called natural antibodies (nAbs), i.e. immunoglobulins generated by B-1 cells, are directed to glycans. nAbs to glycans can be divided in three groups: 1) conservative nAbs, i.e. practically the same in all healthy donors with respect to their epitope specificity and level in blood; 2) allo-antibodies to blood group antigens; 3) plastic antibodies related to the first or the second group but discussed separately because their level changes considerably during diseases and some temporary conditions, in particular inflammation and pregnancy. Antibodies from the third group proved to be prospective markers of a number of diseases, whereas their unusual level (below or above the norm) is not necessarily the consequence of disease/state. Modern microarrays allowed the determination of the human repertoire, which proved to be unexpectedly broad. It was observed that the content of some nAbs reaches about 0.1% of total immunoglobulins. Immunoglobulins of M class dominate for most nAbs, constituting up to 80-90%. Their affinity (to a monovalent glycan, in K _D terms) was found to be within the range 10^?4–10^?6 M. Antibodies to Galβ1-3GlcNAc (Le^C), 4-HSO₃Galβ1-4GalNAc (4′-O-SuLN), Fucα1-3GlcNAc, Fucα1-4GlcNAc, GalNAcα1-3Gal (A_di), Galα1-4Galβ1-4Glc (P^k), Galα1-4Galβ1-4GlcNAc (P₁), GlcNAcα-terminated glycans, and hyaluronic acid should be noted among the nAbs revealed and studied during the last decade. At the same time, a kind of “taboo” is observed for a number of glycans: antibodies to LeX and Le^Y, and almost all gangliosides have not been observed in healthy persons. Many of the revealed nAbs were directed to constrained inner (core) part of glycan, directly adjoined to lipid of cell membrane or protein. The biological function of these nAbs remains unclear; for anti-core antibodies, a role of surveillance on appearance of aberrant, especially cancer, antigens is supposed. The first data related to oncodiagnostics based on quantitation of anti-glycan nAbs are reported.     

Production,characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.     

Contribution of leptin receptor N-linked glycans to leptin binding

The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.     

A functional antibody lacking N-linked glycans is efficiently folded, assembled and secreted by tobacco mesophyll protoplasts

A potential drawback in the use of plants as an expression platform for pharmaceutical proteins such as antibodies is that plant-specific N-glycosylation can result in proteins with altered function and potential antigenicity. In many cases, the N-glycans are essential for the correct folding, assembly and transport of the recombinant proteins. We tested whether progressive removal of glycosylation sites had a detrimental effect on the synthesis, assembly and secretion of a plant-made immunoglobulin G, Guy's 13. Our results indicate that the plant secretory pathway can cope well with aglycosylated antibody chains. The immunoglobulin without N-linked glycans is correctly assembled and secreted by tobacco protoplasts. Capture enzyme-linked immunosorbent assay also shows that antigen-binding properties are unaffected. Our results therefore suggest one possible alternative to the engineering of a humanized glycosylation machinery in plants.     

Cryo-ET of Env on intact HIV virions reveals structural variation and positioning on the Gag lattice

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Novel carbohydrate-binding activity of pancreatic trypsins to N-linked glycans of glycoproteins

How glycosylation affects the reactivity of proteins to trypsin is not well understood. Bovine and porcine pancreatic trypsins were discovered to bind to alpha-Man, Neu5Acalpha2,6Galbeta1,4Glc, and alpha-galactose sequences by binding studies with biotinylated sugar-polymers. Quantitative kinetic studies supported that phenylmethylsulfonyl fluoride (PMSF)-treated trypsin binds to glycolipid analogues possessing alpha-Man or alpha-NeuAc but not to those possessing beta-galactose or beta-GlcNAc residue. Enzyme-linked immunosorbent assay (ELISA) showed that trypsin binds to six kinds of biotinylated glycoproteins possessing high mannose-type and complex-type N-glycans but not to bovine submaxillary mucin, which possesses only O-glycans. Further, the binding of trypsin to glycoproteins was differentially changed by treatments with sequential exoglycosidases, endoglycosidase H, or N-glycosidase F. Quantitative kinetic studies indicated that PMSF-treated trypsin binds with bovine thyroglobulin with the affinity constant of 10(10) m(-1), which was the highest among the glycoproteins examined, and that alpha-galactosidase treatment decreased it to 10(5) m(-1). PMSF-treated trypsin bound to other glycoproteins, including ovomucoid, a trypsin inhibitor, with the affinity constants of 10(8)-10(5) mol(-1) and were markedly changed by glycosidase treatments in manners consistent with the sugar-binding specificities suggested by ELISA. Thus, the binding site for glycans was shown to be distinct from the catalytic site, allowing trypsin to function as an uncompetitive activator in the hydrolysis of a synthetic peptide substrate. Correspondingly the carbohydrate-binding activities of trypsin were unaffected by treatment with PMSF or soybean trypsin inhibitor. The results indicate the presence of an allosteric regulatory site on trypsin that sugar-specifically interacts with glycoproteins in addition to the proteolytic catalytic site.     

Dengue virus infection induces broadly cross-reactive human IgM antibodies that recognize intact virions in humanized BLT-NSG mice

The development of small animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγ^null mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.     

Extension of the in-gel release method for structural analysis of neutral and sialylated N-linked glycans to the analysis of sulfated glycans: application to the glycans from bovine thyroid-stimulating hormone

This paper reports an extension of the in-gel technique for releasing N-linked glycans from glycoproteins for analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry reported by B. Küster, S. F. Wheeler, A. P. Hunter, R. A. Dwek, and D. J. Harvey (1997, Anal. Biochem. 250, 82-101) to allow it to be used for sulfated glycans. The method was used to identify N-linked glycans from bovine thyroid-stimulating hormone. Following glycan release, either in gel or in solution, the glycans were fractionated directly with a porous graphatized carbon column. The sulfated glycans were examined by MALDI mass spectrometry in negative ion mode with d-arabinosazone as the matrix and both neutral and acidic glycans were examined in positive ion mode from 2,5-dihydroxybenzoic acid. Negative ion post-source decay spectra were also obtained. Twenty-two neutral and fifteen sulfated N-linked glycans were identified and it was shown that negligible loss of sulfate groups occurred even though these groups are often readily lost during MALDI analysis. The glycans were mainly sulfated hybrid and biantennary complex structures. Negative ion post-source decay and positive ion collision-induced fragmentation spectra were obtained.     

Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs)

The crystal structures of the glycosylated N-terminal two domains of ICAM-1 and ICAM-2 provided a framework for understanding the role of glycosylation in the structure and function of intercellular adhesion molecules (ICAMs). The most conserved glycans were less flexible in the structures, interacting with protein residues and contributing to receptor folding and expression. The first N-linked glycan in ICAM-2 contacts an exposed tryptophan residue, defining a conserved glycan-W motif critical for the conformation of the integrin binding domain. The absence of this motif in human ICAM-1 exposes regions used in receptor dimerization and rhinovirus recognition. Experiments with soluble molecules having the N-terminal two domains of human ICAMs identified glycans of the high mannose type N-linked to the second domain of the dendritic cell-specific ICAM-grabbing nonintegrin lectin-ligands ICAM-2 and ICAM-3. About 40% of those receptor molecules bear endoglycosidase H sensitive glycans responsible of the lectin binding activity. High mannose glycans were absent in ICAM-1, which did not bind to the lectin, but they appeared in ICAM-1 mutants with additional N-linked glycosylation and lectin binding activity. N-Linked glycosylation regulate both conformation and immune related functions of ICAM receptors.     

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.     

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.     

Sulfated N-linked oligosaccharides in mammalian cells. II. Identification of glycosaminoglycan-like chains attached to complex-type glycans

In the preceding paper (Roux, L., Holojda, S., Sundblad, G., Freeze, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889) we described the metabolic labeling and isolation of sulfated N-linked oligosaccharides from mammalian cell lines. All cell lines studied contained a class of sulfated sialylated complex-type chains with 2-6 negative charges. In this paper, we show that bovine pulmonary arterial endothelial (CPAE) and human erythroleukemia (K562) cell lines also contain a class of more highly charged sulfated but less sialylated oligosaccharides. These molecules were further characterized by ion exchange chromatography and various enzymatic and chemical treatments. In both cell lines they contained greater than 6 negative charges, but those from K562 were even more highly charged than those from CPAE. Nitrous acid, heparinase, and heparitinase degradation of K562 oligosaccharides released 88, 64, and 78%, respectively, of 35S label. Combined digestion with the two enzymes resulted in 87% release. The corresponding values for CPAE were 48, 25, and 50% (60% for the two enzymes together). Chondroitinase ABC (or AC) digestion of K562 and CPAE oligosaccharides released 10 and 5%, respectively. About 30% of the 35S-labeled oligosaccharides from CPAE were sensitive to endo-beta-galactosidase, indicating that poly-N-acetyl-lactosamine structures were present on some chains. Highly charged [3H]mannose-labeled sulfated oligosaccharides from CPAE cells became neutral after treatment with heparinase/heparitinase but were resistant to Pronase, further proving that glycosaminoglycan (GAG)-like chains were directly attached to N-linked oligosaccharides. Such neutralized oligosaccharides did not bind to concanavalin A-Sepharose, but some interacted with phytohemagglutinin L4, indicating that they were bi-, tri-, or tetra-antennary complex-type chains. Thus, K562 and CPAE cells contain different types of GAG chains directly attached to asparagine-linked oligosaccharides. Such molecules were not found in many other cell lines that synthesize the more typical O-linked GAG chains. This suggests that the occurrence of these novel N-linked chains is not a random event resulting from accidental initiation of GAG chain synthesis on N-linked intermediates in the Golgi apparatus.     

Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env

We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1_AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.     

A monoclonal antibody to a cross-reactive idiotype on cationic human anti-DNA antibodies expressing lambda light chains: a new reagent to identify a potentially differential pathogenic subset

Anti-double-stranded DNA antibodies are commonly found in the serum of patients with systemic lupus erythematosus (SLE). They are a heterogeneous group of antibodies thought to differ in pathogenicity. The degree of heterogeneity and the structural correlates of pathogenicity, however, remain poorly defined. To address these questions we have been generating anti-idiotypic antibodies to the anti-DNA antibodies found in the serum of SLE patients. In this paper we report the generation and characterization of a new murine monoclonal anti-idiotype, 8.12, that recognizes a subset of anti-DNA antibodies that is present in serum of approximately 50% of patients with SLE. The 8.12 anti-idiotype recognizes uniquely cationic anti-DNA antibodies, all of which express lambda light chains. In murine models of SLE, it has been suggested that cationic anti-DNA antibodies are preferentially deposited in the kidney. It may be, therefore, that 8.12 recognizes a subset of anti-DNA antibodies of particular pathogenic significance.     

Activation of murine CD4+ and CD8+ T lymphocytes leads to dramatic remodeling of N-linked glycans

Differentiation and activation of lymphocytes are documented to result in changes in glycosylation associated with biologically important consequences. In this report, we have systematically examined global changes in N-linked glycosylation following activation of murine CD4 T cells, CD8 T cells, and B cells by MALDI-TOF mass spectrometry profiling, and investigated the molecular basis for those changes by assessing alterations in the expression of glycan transferase genes. Surprisingly, the major change observed in activated CD4 and CD8 T cells was a dramatic reduction of sialylated biantennary N-glycans carrying the terminal NeuGcalpha2-6Gal sequence, and a corresponding increase in glycans carrying the Galalpha1-3Gal sequence. This change was accounted for by a decrease in the expression of the sialyltransferase ST6Gal I, and an increase in the expression of the galactosyltransferase, alpha1-3GalT. Conversely, in B cells no change in terminal sialylation of N-linked glycans was evident, and the expression of the same two glycosyltransferases was increased and decreased, respectively. The results have implications for differential recognition of activated and unactivated T cells by dendritic cells and B cells expressing glycan-binding proteins that recognize terminal sequences of N-linked glycans.     

Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner

Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V_H region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG₁ antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Differential binding of cross-reactive anti-DNA antibodies to mesangial cells: the role of alpha-actinin

Target Ag display is a necessary requirement for the expression of certain immune-mediated kidney diseases. We previously had shown that anti-DNA Abs that cross-react with alpha-actinin may be important in the pathogenesis of murine and human lupus nephritis; in murine models, we had found that a significant proportion of pathogenic serum and kidney-deposited Igs are alpha-actinin reactive. Furthermore, a pathogenic anti-DNA/alpha-actinin Ab showed enhanced binding to immortalized mesangial cells (MCs) derived from a lupus prone MRL-lpr/lpr mouse as compared with MCs from BALB/c mice which are not susceptible to spontaneous lupus, suggesting that kidney alpha-actinin expression may be contributing to nephritis. In the current study, we established that two isoforms of alpha-actinin that are present in the kidney, alpha-actinin 1 and alpha-actinin 4, can both be targeted by anti-alpha-actinin Abs. We found novel sequence polymorphisms between MRL-lpr/lpr and BALB/c in the gene for alpha-actinin 4. Moreover, alpha-actinin 4 and a splice variant of alpha-actinin 1 were both expressed at significantly higher levels (mRNA and protein) in MCs from the lupus prone MRL-lpr/lpr strain. Significantly, we were able to confirm these differences in intact kidney by examining glomerular Ig deposition of anti-alpha-actinin Abs. We conclude that enhanced alpha-actinin expression may determine the extent of Ig deposition in the Ab-mediated kidney disease in lupus. Modulation of Ag expression may be a promising approach to down-regulate immune complex formation in the target organ in individuals with circulating pathogenic Abs.