While the induction of a neutralizing antibody response against HIV remains a daunting goal, data from both natural infection and vaccine-induced immune responses suggest that it may be possible to induce antibodies with enhanced Fc effector activity and improved antiviral control via vaccination. However, the specific features of naturally induced HIV-specific antibodies that allow for the potent recruitment of antiviral activity and the means by which these functions are regulated are poorly defined. Because antibody effector functions are critically dependent on antibody Fc domain glycosylation, we aimed to define the natural glycoforms associated with robust Fc-mediated antiviral activity. We demonstrate that spontaneous control of HIV and improved antiviral activity are associated with a dramatic shift in the global antibody-glycosylation profile toward agalactosylated glycoforms. HIV-specific antibodies exhibited an even greater frequency of agalactosylated, afucosylated, and asialylated glycans. These glycoforms were associated with enhanced Fc-mediated reduction of viral replication and enhanced Fc receptor binding and were consistent with transcriptional profiling of glycosyltransferases in peripheral B cells. These data suggest that B cell programs tune antibody glycosylation actively in an antigen-specific manner, potentially contributing to antiviral control during HIV infection.

Objective To examine the association of IgG galactosylation aberrancy with disease parameters in rheumatoid arthritis (RA). Methods N-glycan analysis of serum from multiple cohorts was performed. IgG N-glycan content and timing of N-glycan aberrancy relative to disease onset was compared in healthy and RA subjects. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, gender, age, anti-CCP titer, disease duration, and CRP on aberrant galactosylation was determined using multivariate analysis. N-glycan content was also compared between epitope affinity purified autoantibodies and the remaining repertoire IgG in RA subjects. Results Our results confirm the aberrant galactosylation of IgG in RA (1.36 ± 0.43) compared to healthy controls (1.01 ± 0.23) (P < 0.0001). We observe a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman rho = 0.37, p<0.0001). This correlation is higher in females [Spearman rho = 0.60 (P<0.0001)] than males [Spearman rho = 0.16 (P = 0.10)]. Further, IgG galactosylation aberrancy substantially predates onset of arthritis and the diagnosis of RA (3.5 years) and resides selectively in the anti-citrullinated peptide autoantibody fraction. Conclusions Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, that associates with disease activity in a gender specific manner, and that resides preferentially in autoantibodies.

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The majority of proteins present in human serum/plasma are glycoproteins, validating this fluid as an ideal starting material for N-glycan analysis and discovery of potential biomarkers. The glycoprotein content for both serum and plasma is very similar, except for proteins removed in the coagulation process, including fibrinogen. Our aim was to characterize fibrinogen glycosylation in order to determine its contribution to differences between serum and plasma N-glycomes. N-Glycans from human fibrinogen were released, labeled, and analyzed by HILIC-HPLC and MS. Structural characterization of fibrinogen subunits revealed that the α chain was not N-glycosylated, whereas β and γ contained identical oligosaccharide structures, mainly biantennary digalactosylated monosialylated structures (A2G2S1) and biantennary digalactosylated disialylated structures (A2G2S2). Blood was collected from five healthy volunteers into four testing tubes: silicone-coated glass for serum and EDTA, Na-heparin, and Li-heparin glass tubes for plasma. N-Glycans were analyzed using the high-throughput HILIC-HPLC method. N-Glycan profiles from serum and plasma samples differed largely in glycans identified in fibrinogen, suggesting that this glycoprotein represents a major factor distinguishing these body fluids. This result emphasizes the important of consistent body fluid collection practices in biomarker discovery studies.

Skp1 is an adaptor-like protein in E3SCF -ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Gal␣1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn 2؉ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Gal␣1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to ␣1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K m and 35-fold higher V max compared with a simple ␣-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V max advantage, but still much less active than Skp1 itself because of a K m difference. These findings indicate that ␣-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Gal␣1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

A unique "enzyme catalytic promiscuity" has recently been observed, wherein a phosphodiester and a phosphonate ester are hydrolyzed by a dinuclear aminopeptidase and its metal derivatives from Streptomyces griseus (SgAP) [Park, H. I., Ming, L.-J. (1999) Angew. Chem., Int. Ed. Engl. 38, 2914-2916 and Ercan, A., Park, H. I., Ming, L.-J. (2000) Chem. Commun. 2501-2502]. Because tetrahedral phosphocenters often serve as transition-state inhibitors toward the hydrolysis of the peptide, phosphoester hydrolysis by peptidases is thus not expected to occur effectively and must take place through a unique mechanism. Owing to the very different structures and mechanistic requirements between phosphoesters and peptides during hydrolysis, the study of this effective phosphodiester hydrolysis by SgAP may provide further insight into the action of this enzyme that is otherwise not obtainable from regular peptide substrates. We present herein a detailed investigation of both peptide and phosphodiester hydrolyses catalyzed by SgAP. The latter exhibits a first-order rate enhancement of 4 x 10(10)-fold compared to the uncatalyzed reaction at pH 7.0 and 25 degrees C. The results suggest that peptide and phosphodiester hydrolyses by SgAP may share a common reaction mechanism to a certain extent. However, their differences in pH dependence, phosphate and fluoride inhibition patterns, and proton inventory reflect that they must follow different pathways. Mechanisms for the two hydrolyses are drawn on the basis of the results, which provide the foundation for further investigation of the catalytic promiscuity of this enzyme by means of physical and molecular biology methods. The catalytic versatility of SgAP suggests that this enzyme may serve as a unique "natural model system" for further investigation of dinuclear hydrolysis. A better understanding of enzyme catalytic promiscuity is also expected to shed light on the evolution and action of enzymes.

IntroductionRheumatoid arthritis (RA) is associated with hypogalactosylation of immunoglobulin G (IgG). We examined whether a proxy measure for galactosylation of IgG N-glycans could predict response to therapy or was differentially affected by methotrexate (MTX) or TNF blockade.MethodsUsing a previously defined normal phase high-performance liquid chromatography approach, we ascertained the galactosylation status of whole serum N-glycans in two well-defined RA clinical cohorts: the Autoimmune Biomarkers Collaborative Network (n = 98) and Nested I (n = 64). The ratio of agalactosylated to monogalactosylated N-glycans in serum (sG0/G1) was determined before and during therapy with MTX or TNF inhibition and correlated with anticitrullinated peptide antibody (ACPA) status and clinical response as assessed by 28-joint Disease Activity Score utilizing C-reactive peptide and European League Against Rheumatism response criteria.ResultsRA patients from both cohorts exhibited elevation of sG0/G1 at baseline. Improvement in clinical scores correlated with a reduction in sG0/G1 (Spearman's ρ = 0.31 to 0.37; P < 0.05 for each cohort). However, pretreatment sG0/G1 was not predictive of clinical response. Changes in sG0/G1 were similar in the MTX and TNF inhibitor groups. Corrected for disease activity, ACPA positivity correlated with higher sG0/G1.ConclusionsBaseline serum N-glycan hypogalactosylation, an index previously correlated with hypogalactosylation of IgG N-glycans, did not distinguish patients with rheumatoid arthritis who were likely to experience a favorable clinical response to MTX or TNF blockade. Clinical improvement was associated with partial glycan normalization. ACPA-positive patients demonstrated enhanced N-glycan aberrancy compared with ACPA-negative patients.