Galactosylation Of IgG Research Articles

Abnormal N-glycan fucosylation, galactosylation, and sialylation of IgG in adults with classical galactosemia, influence of dietary galactose intake.

BackgroundClassical galactosemia (CG) (OMIM #230400) is a rare disorder of carbohydrate metabolism, due to deficiency of galactose‐1‐phosphate uridyltransferase (EC 2.7.7.12). The pathophysiology of the long‐term complications, mainly cognitive, neurological, and female infertility remains poorly understood.ObjectivesThis study investigated (a) the association between specific IgG N‐glycosylation biomarkers (glycan peaks and grouped traits) and CG patients (n = 95) identified from the GalNet Network, using hydrophilic interaction ultraperformance liquid chromatography and (b) a further analysis of a GALT c.563A‐G/p.Gln188Arg homozygous cohort (n = 49) with correlation with glycan features with patient Full Scale Intelligence Quotient (FSIQ), and (c) with galactose intake.ResultsA very significant decrease in galactosylation and sialylation and an increase in core fucosylation was noted in CG patients vs controls (P < .005). Bisected glycans were decreased in the severe GALT c.563A‐G/p.Gln188Arg homozygous cohort (n = 49) (P < .05). Logistic regression models incorporating IgG glycan traits distinguished CG patients from controls. Incremental dietary galactose intake correlated positively with FSIQ for the p.Gln188Arg homozygous CG cohort (P < .005) for a dietary galactose intake of 500 to 1000 mg/d. Significant improvements in profiles with increased galactose intake were noted for monosialylated, monogalactosylated, and monoantennary glycans.ConclusionThese results suggest that N‐glycosylation abnormalities persist in CG patients on dietary galactose restriction which may be modifiable to a degree by dietary galactose intake.

Quantitative analysis of serum-based IgG agalactosylation for tuberculosis auxiliary diagnosis.

Tuberculosis (TB) is the leading infectious cause of mortality worldwide, especially in developing countries. However, effective means for TB diagnosis, especially for bacillus-negative (Bn) TB laboratory diagnosis, are urgently needed. In the present study, serum IgG from each tuberculosis patients and healthy controls was purified using affinity chromatography. The samples were then analyzed using mass spectrometry (MS) and ultraperformance liquid chromatography (UPLC) methods. We quantitatively assessed the changes of serum IgG galactosylation in 567 human serum samples including 377 pulmonary TB patients and 190 healthy donors (HDs). We found significantly more agalactosylated (G0) vs monogalactosylated (G1) and digalactosylated (G2) N-glycans of IgG in TB patients, including smear-negative TB patients, than in HDs. The detection rate of TB diagnostic performance by MS for IgG-Gal ratio G0/(G1+G2×2) is 90.48% for bacillus-positive (Bp) and 73.16% for Bn TB patients. Further, combination of MS method with other routine laboratory TB diagnostic methods significantly increased the detection rate to 91.01%-98.39%. Similar results were observed in Mycobacterium tuberculosis (M. tb) infection mouse models. The decrease in galactosylation of IgG in TB patients was also qualitatively confirmed using specific lectin blot assay. Using the above techniques, we can discriminate the content of IgG G0 with terminal N-acetylglucosamine and IgG-Gal ratio G0/(G1+G2×2) between TB patients and HDs. Our data suggest that quantitative analysis of serum-based IgG-Gal ratio G0/(G1+G2×2) could be used for TB auxiliary diagnosis with high effectiveness and feasibility and its combination with other routine laboratory TB diagnostic methods could remarkably improve the detection rate.

The Self-Administered Rheumatoid Arthritis Disease Activity Index (RADAI) is Less Accurate than DAS28-CRP as a Disease Activity Measure in Pregnancy.

ObjectiveDisease Activity Score 28 (DAS28)–using the C‐reactive protein (CRP) level has been validated to determine disease activity in rheumatoid arthritis (RA) patients during pregnancy. A self‐administered questionnaire like Rheumatoid Arthritis Disease Activity Index (RADAI) has practical advantages over DAS28‐CRP, and in this study, we aimed to validate the RADAI for use during pregnancy.MethodsPatients were derived from a prospective cohort on RA and pregnancy (Pregnancy‐induced Amelioration of Rheumatoid Arthritis study). To validate the RADAI as a disease activity measure, the disease course over time and the disease activity states were compared with the DAS28‐CRP. Furthermore, the RADAI was compared with DAS28‐CRP in predicting fertility and pregnancy outcomes. Finally, to test construct validity, correlation of both RADAI and DAS28‐CRP with a biomarker (galactosylation of immunoglobulin G [IgG]) were determined and compared.ResultsIn total, 269 patients were analyzed in this study. Mean RADAI scores showed a great decline in disease activity in the first trimester compared with DAS28‐CRP (mean RADAI, −1.13; mean, DAS28‐CRP, −0.04). Correlations between DAS28‐CRP and RADAI scores were moderate to good (0.44 < ρ < 0.71). Agreement in disease states was low (0.26 < κ < 0.51). Time to pregnancy was different between disease states according to DAS28‐CRP (P = 0.03), but not according to RADAI (P = 0.56). Only DAS28‐CRP could predict birthweight (DAS28‐CRP β‐0.17, P = 0.04; RADAI β‐0.09, P = 0.10). Both DAS28‐CRP and RADAI were associated with galactosylation of IgG at specific time points, but only change in DAS28‐CRP was correlated with change in galactosylation of IgG from preconception to pregnancy.ConclusionThe RADAI could not be validated as a disease activity measure during pregnancy. DAS28‐CRP remains the gold standard of measuring disease activity in pregnancy.

Nickel and cobalt affect galactosylation of recombinant IgG expressed in CHO cells.

Glycosylation is an important product quality attribute of antibody biopharmaceuticals. It involves enzymatic addition of oligosaccharides on proteins by sequential action of glycosyltransferases and glycosidases in the endoplasmic reticulum and golgi. Some of these enzymes like galactosyltransferase and N-acetylglucosaminyltransferase-I require trace metal cofactors. Variations in trace metal availability during production can thus affect glycosylation of recombinant glycoproteins such as monoclonal antibodies. Variability in trace metal concentrations can be introduced at multiple stages during production such as due to impurities in raw materials for culture medium and leachables from bioreactors. Knowledge of the effect of various trace metals on glycosylation can help in root-cause analysis of unintended variability in glycosylation. In this study, we investigated the effect of nickel and cobalt on glycosylation of recombinant IgG expressed in Chinese hamster ovary cells. Nickel concentrations below 500µM did not affect glycosylation, but above 500µM it significantly decreases galactosylation of IgG. Cobalt at 50µM concentration causes slight increase in G1F glycans (mono galactosylated) as previously reported. However, higher concentrations result in a small increase in G0F (non galactosylated) glycans. This effect of nickel and cobalt on galactosylation of recombinant IgG can be reversed by supplementation of uridine and galactose which are precursors to UDP-Galactose, a substrate for the enzymatic galactosylation reaction.

Zinc supplementation decreases galactosylation of recombinant IgG in CHO cells.

Trace element composition of culture medium can be altered to modulate glycoform of recombinant glycoproteins. In this study, we show that Zn2+ supplementation at or above 100μM decreases galactosylation of recombinant IgG expressed in Chinese Hamster Ovary cells. This decrease in galactosylation is not due to reduced galactosyltransferase expression. This effect persists upon supplementation of galactose and uridine to the culture, indicating that it may not be due to reduced UDP-Gal availability. Measurements of galactosyltransferase activity in the cell lysate show that activity decreases with increasing Zn2+/Mn2+ ratio. This suggests that one possible explanation of the effect of Zn2+ may be reduced intracellular galactosyltransferase activity due to increase in Zn2+/Mn2+ ratio. Consistent with this, the decrease in galactosylation of IgG could be reversed by supplementation of Mn2+ (a cofactor of galactosyltransferase) which increases intracellular Mn2+ content. Measurement of total intracellular Zn2+ content, however, indicates no significant upregulation of total intracellular Zn2+ content and no significant downregulation of intracellular Mn2+ content with Zn2+ supplementation. One possible explanation could be that cellular detoxification response to higher extracellular Zn2+ concentration might lead to changes in intracellular distribution of Mn2+. In this case, Zn2+ supplementation would be expected to interfere with other known effects of Mn2+. Indeed, the previously reported increase in high mannose glycans upon Mn2+ supplementation in the absence of glucose is reversed by Zn2+ supplementation. This study also suggests the use of Mn2+ supplementation as a strategy to overcome the effect of lot-to-lot variability in trace element concentrations on galactosylation.

ACPA IgG galactosylation associates with disease activity in pregnant patients with rheumatoid arthritis

ObjectivesPatients with autoantibody-positive rheumatoid arthritis (RA) are less likely to experience pregnancy-induced improvement of RA disease activity (DAS28-C reactive protein (CRP)) compared with patients with autoantibody-negative RA. Anti-citrullinated protein antibodies...

Conserved FcγR- glycan discriminates between fucosylated and afucosylated IgG in humans and mice

The binding strength between IgG and FcγR is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human FcγRIIIa up to ∼20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to ∼40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate-carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFcγRIIIa and hFcγRIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse FcγR, mFcγRIV. The N162-glycan in mFcγRIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFcγRIIIa. However, unlike hFcγRIIIa, mFcγRIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human FcγRIII family and its orthologous mouse FcγRIV to be functionally conserved.

Changes of glycosylation of IgG in rheumatoid arthritis patients treated with methotrexate

PurposeIn patients with active rheumatoid arthritis (RA) decrease of galactosylation is correlated with disease activity. The aim of our study was to evaluate an effect of methotrexate therapy on glycosylation disturbances of IgG in RA patients. Materials/methodsIgG glycosylation in 40 patients with active RA treated with methotrexate for 12 months prior to and after treatment were compared. The control group consisted of 20 healthy volunteers. IgG glycosylation was assessed using biotinylated lectins and immunosorbent ELISA assay. For galactose specificity Datura stramonium lectin (DSA), for sialic acid Sambucus nigra (SNA) and Maackia amurensis (MAA) and for fucose residue Areulia auranta (AAA) lectins were used. ResultsIn RA-cases N-glycan galactosylation and sialylation of IgG before treatment were significantly lower than in healthy subjects (for DSA, MAA lectins p<0.001 and SNA p<0.05). Significant increase of IgG galactosylation and sialylation in RA patients after therapy (for DSA, MAA and SNA lectin p<0.05) was detected. Moreover the glycosylation disturbances of N-glycan IgG were strongly associated with changes of disease activity based on disease activity score. For fucose residues significantly higher absorbency of AAA lectin in RA patients before treatment was observed compared to control subjects (p<0.05) and slightly, not significantly decreased after MTX therapy. ConclusionsDefect of galactosylation of IgG in RA patients is a useful marker of disease activity that may be used for the assessment of therapy effectiveness. The role of IgG fucosylation and sialylation in RA pathogenesis has still to be determined.

Filariasis asymptomatically infected donors have lower levels of disialylated IgG compared to endemic normals.

Helminths induce strong regulatory and T helper 2-type responses, whereby antibody-derived host protection and regulation are essential components. Lymphatic filariasis is an immune-mediated spectral disease that manifests as two main clinical outcomes: chronic pathology or asymptomatic infection. These outcomes depend on a multitude of factors, including parasite-induced immunoregulation and host genetic background; antibody responses contribute to this outcome. N-glycosylation of the Fc region of antibodies is a post-translational modification required for the structure and molecular function, influencing host inflammatory and regulatory responses. Altered IgG glycosylation correlates with disease, whereby decreased galactosylation is associated with inflammation while increased sialylation is associated with anti-inflammatory responses. We purified N-linked glycans from the Fc region of total IgG from Wuchereria bancrofti-infected patients characterizing the two clinical manifestations (chronic pathology and asymptomatic infection) and compared them to infection-free endemic normals. Using capillary electrophoresis, we found that there was no difference in galactosylation of total IgG between the three groups; however, asymptomatically infected patients had significantly lower levels of disialylated IgG compared to endemic normals and patients with pathology. These data suggest that while galactosylation does not contribute to disease outcome, sialylation may be involved in asymptomatic infection.

The role of glycosylation in IBD.

A number of genetic and immunological studies give impetus for investigating the role of glycosylation in IBD. Experimental mouse models have helped to delineate the role of glycosylation in intestinal mucins and to explore the putative pathogenic role of glycosylation in colitis. These experiments have been extended to human studies investigating the glycosylation patterns of intestinal mucins as well as levels of glycans of serum glycoproteins and expression of glycan receptors. These early human studies have generated interesting hypotheses regarding the pathogenic role of glycans in IBD, but have generally been restricted to fairly small underpowered studies. Decreased glycosylation has been observed in the intestinal mucus of patients with IBD, suggesting that a defective inner mucus layer might lead to increased bacterial contact with the epithelium, potentially triggering inflammation. In sera, decreased galactosylation of IgG has been suggested as a diagnostic marker for IBD. Advances in glycoprofiling technology make it technically feasible and affordable to perform high-throughput glycan pattern analyses and to build on previous work investigating a much wider range of glycan parameters in large numbers of patients.

AB0121 Changes of Glycosylation of Immunoglobulin G in Rheumatoid Arthritis Patients Treated with Methotrexate

BackgroundIn patients with active rheumatoid arthritis (RA) decrease of galactosylation of IgG is observed. It has been shown that agalactosylation is correlated with disease activity (1, 3, 4).ObjectivesThe aim of...

AB0945 Effect of adalimumab treatment of psoriatic arthritis patients (PSA) on galactosylation of serum IGG. A preliminary report

BackgroundThe first reports on the differences in glycosylation, which may negatively affect the antibody function, came from the studies on rheumatoid arthritis (RA), and specifically, the occurrence of glycoform G0...

IgG Glycosylation Changes andMBL2Polymorphisms: Associations with Markers of Systemic Inflammation and Joint Destruction in Rheumatoid Arthritis

To examine whether IgG glycosylation changes and MBL2 genotypes are associated with systemic inflammation and joint destruction in rheumatoid arthritis (RA). IgG N-glycan content was determined from serum in 118 patients with RA by high-throughput glycan analysis using normal-phase high-pressure liquid chromatography. MBL2 extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) were determined. Systemic inflammation was assessed by serum levels of C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). Joint destruction was assessed by total Sharp score (TSS) and alloplastic surgery records. IgG hypogalactosylation was significantly correlated to IL-6 (Spearman's rho = 0.32, p < 0.001), CRP (Spearman's rho = 0.31, p < 0.001), TSS (Spearman's rho = 0.25, p = 0.01), and alloplastic replacement of joints (Spearman's rho = 0.18, p = 0.05). In multivariate analysis including age, CRP, anticitrullinated protein antibodies (ACPA), and other confounders, IgG hypogalactosylation was significantly associated with TSS (p = 0.014) and alloplastic joint replacement (OR 76.5, p = 0.041) in patients homozygous for the high expression MBL2 genotype YA/YA, but not in carriers of lower expression genotypes. Decreased galactosylation of IgG correlated to markers of inflammation, i.e., IL-6 and CRP. Only in patients homozygous for high expression of the MBL2 genotype YA/YA was IgG hypogalactosylation associated with markers of joint destruction. Our results suggest that inflammation-associated decreased galactosylation of IgG combined with high expression MBL2 genotypes are involved in the pathophysiology of RA.

IgG N-glycans as potential biomarkers for determining galactose tolerance in Classical Galactosaemia

N-glycan processing and assembly defects have been demonstrated in untreated and partially treated patients with Classical Galactosaemia. These defects may contribute to the ongoing pathophysiology of this disease. The aim of this study was to develop an informative method of studying differential galactose tolerance levels and diet control in individuals with Galactosaemia, compared to the standard biochemical markers. Ten Galactosaemia adults with normal intellectual outcomes were analyzed in the study. Five subjects followed galactose liberalization, increments of 300 mg to 4000 mg/day over 16 weeks, and were compared to five adult Galactosaemia controls on a galactose restricted diet. All study subjects underwent clinical and biochemical monitoring of red blood cell galactose-1-phosphate (RBC Gal-1-P) and urinary galactitol levels. Serum N-glycans were isolated and analyzed by normal phase high-performance liquid chromatography (NP-HPLC) with galactosylation of IgG used as a specific biomarker of galactose tolerance. IgG N-glycan profiles showed consistent individual alterations in response to diet liberalization. The individual profiles were improved for all, but one study subject, at a galactose intake of 1000 mg/day, with decreases in agalactosylated (G0) and increases in digalactosylated (G2) N-glycans. We conclude that IgG N-glycan profiling is an improved method of monitoring variable galactosylation and determining individual galactose tolerance in Galactosaemia compared to the standard methods.

Substitution of glutamine by glutamate enhances production and galactosylation of recombinant IgG in Chinese hamster ovary cells

The effect of ammonia on Chinese hamster ovary (CHO) cell growth and galactosylation of recombinant immunoglobulin (rIgG) was investigated using shaking flasks with serum free media containing 0-15 mM NH(4)Cl. The elevated ammonia inhibited cell growth and negatively affected the galactosylation of rIgG. At 15 mM NH(4)Cl, the proportions of monogalactosylated glycan with fucosex (monogalactosylated glycan with fucose) and digalactosylated glycan with fucose (G2F) were 23.9% and 6.3% lower than those at 0 mM NH(4)Cl, respectively. To reduce ammonia formation by cells, glutamate was examined as a substitute for glutamine. The use of glutamate reduced the accumulation of ammonia and enhanced the production of rIgG while depressing cell growth. At 6 mM glutamate, ammonia level did not exceed 2 mM, which is only one third of that at 6 mM glutamine. Also, a 1.7-fold increase in the titer of rIgG and specific rIgG productivity, q (rIgG), was achieved at 6 mM glutamate. The galactosylation of rIgG was favorable at 6 mM glutamate. The proportion of galactosylated glycans, G1F and G2F, at 6 mM glutamate was 59.8%, but it was 50.4% at 6 mM glutamine. The use of glutamate also increased complement-dependent cytotoxicity activity, one of the effector functions of rIgG. Taken together, substitution of glutamine by glutamate can be considered relevant for the production of rIgG in CHO cells since glutamate not only enhances q (rIgG) but also generates a higher galactosylation essential for the effector function of rIgG.

Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis.

To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA). Analysis of N-glycan in serum samples from multiple cohorts was performed. The IgG N-glycan content and the timing of N-glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anti-cyclic citrullinated peptide (anti-CCP) antibody titer, disease duration, and C-reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N-glycan content was also compared between epitope affinity-purified autoantibodies and the remaining IgG repertoire in RA patients. Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean +/- SD 1.36 +/- 0.43 versus 1.01 +/- 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's rho = 0.37, P < 0.0001). This correlation was higher in women (Spearman's rho = 0.60, P < 0.0001) than in men (Spearman's rho = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction. Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex-specific manner, and resides preferentially in autoantibodies.

Glycosylation Changes on Serum Glycoproteins in Ovarian Cancer May Contribute to Disease Pathogenesis

Ovarian cancer is the most lethal of all gynaecological cancers among women. Serum CA125 is the only biomarker that is used routinely and there is a need for further complementary biomarkers both in terms of sensitivity and specificity. N-glycosylation changes in ovarian cancer serum glycoproteins include a decrease in galactosylation of IgG and an increase in sialyl Lewis X (SLex) on haptoglobin β-chain, α1-acid glycoprotein and α1-antichymotrypsin. These changes are also present in chronic inflammation but not in malignant melanoma, where there are low levels of inflammatory processes. Acute phase proteins carrying increased amounts of SLex have an increased half-life. Sialylation of acute phase proteins also decreases apoptosis favouring survival of cancer cells. Cancer cells produce inflammatory cytokines which influence glycosylation processing in liver parenchymal cells. Altered glycosylation of the acute phase protein transferrin plays an important role in iron homeostasis. Glycosylated transferrin and its glycans have anti-apoptotic properties and many transferrin receptors in carcinoma could play a role in development of anaemia. Decreased galactosylation and sialylation of IgG increases the cytotoxicity of natural killer cells and complement activation via mannose-binding lectin (MBL). Altered glycosylation of acute phase proteins and IgG suggests that cancer regulates certain pathways favouring cancer cells survival.

Clinical images: Three‐dimensional computed tomography imaging of tophaceous gout

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Galactosylation of IgG from rheumatoid arthritis (RA) patients – changes during therapy

It is well documented that serum IgG from rheumatoid arthritis (RA) patients exhibits decreased galactosylation of its conservative N-glycans (Asn-297) in CH2 domains of the heavy chains; it has been shown that this agalactosylation is proportional to disease severity. In the present investigation we analyzed galactosylation of IgG derived from the patients using a modified ELISA-plate test, biosensor BIAcore and total sugar analysis (GC-MS). For ELISA and BIAcore the binding of IgG preparations, purified from the patients' sera, to two lectins: Ricinus communis (RCA-I) and Griffonia simplicifolia (GSL-II) was applied. Based on ELISA-plate test an agalactosylation factor (AF, a relative ratio of GSL-II/RCA-I binding) was calculated, which was proportional to actual disease severity. Repeated testing of several patients before and after treatment with methotrexate (MTX) alone or in combination with Remicade (a chimeric antibody anti-TNF-alpha) supplied results indicating an increase of IgG galactosylation during the treatment. This introductory observation suggests that IgG galactosylation may be an additional indicator of the RA patients' improvement.

Increased levels of galactose-deficient IgG in sera of HIV-1-infected individuals

The IgG from sera of patients with chronic inflammatory diseases of autoimmune character or some chronic microbial infections is frequently deficient in galactose on N-linked glycans. However, this phenomenon has not been investigated at length in human viral infections. To evaluate the glycosylation of serum IgG in HIV-1-positive patients. Psathyrella velutina lectin was used in enzyme-linked immunosorbent and Western blot assays to determine glycosylation. In addition, gas-liquid chromatography and mass spectrometry were utilized to confirm the galactose deficiency observed in the lectin-binding assays. HIV-1-infected individuals had significantly higher levels of galactose-deficient IgG than healthy controls. In fact, the galactose deficiency of the N-linked glycans observed in other diseases was even more profound in HIV-1 infection. This deficiency was primarily restricted to IgG when total serum glycoproteins were evaluated and IgG1 was the subclass most affected in all patients. Also, a significant increase in lectin binding was observed on IgG2 and IgG4 from HIV-1-positive females compared with HIV-1-negative females. Identification of deficient galactosylation of serum IgG from HIV-1-infected patients extended the spectrum of diseases in which this phenomenon has been observed. In addition, the results suggest yet another aspect of immune dysfunction as a result of HIV-1 infection.