26 research outputs found
Divergent Behavior of Glycosylated Threonine and Serine Derivatives in Solid Phase Peptide Synthesis
Solid phase peptide coupling of glycosylated threonine derivatives was systematically evaluated. In contrast to glycosylated serine derivatives which are highly prone to epimerization, glycosylated threonine derivatives produce only negligible amounts of epimerization. Under forcing conditions, glycosylated threonine analogs undergo β-elimination, rather than epimerization. Mechanistic studies and molecular modeling were used to understand the origin of the differences in reactivity
Competition between Serum IgG, IgM, and IgA Anti-Glycan Antibodies
<div><p>Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies.</p></div
GalNAc-Tyrosine Is a Ligand of Plant Lectins, Antibodies, and Human and Murine Macrophage Galactose-Type Lectins
In
2011, a new type of protein <i>O</i>-glycosylation
was discovered in which <i>N</i>-acetylgalactosamine is
attached to the side chain of tyrosine (GalNAc-Tyr). While present
on dozens of proteins, the biological roles of GalNAc-Tyr are unknown.
To gain insight into this new type of modification, we synthesized
a group of GalNAc-Tyr glycopeptides, constructed microarrays, and
evaluated potential recognition of GalNAc-Tyr by a series of glycan-binding
proteins. Through a series of >150 microarray experiments, we assessed
binding properties of a variety of plant lectins, monoclonal antibodies,
and endogenous lectins. VVL, HPA, and SBA were all found to bind tightly
to GalNAc-Tyr, and several Tn binding antibodies and blood group A
antibodies were found to cross-react with GalNAc-Tyr. Thus, detection
of GalNAc-Tyr modified proteins is an important consideration when
analyzing results from these reagents. Additionally, we evaluated
potential recognition by two mammalian lectins, human (hMGL) and murine
(mMGL-2) macrophage galactose type C-type lectins. Both hMGL and mMGL-2
bound tightly to GalNAc-Tyr determinants. The apparent <i>K</i><sub>d</sub> values (∼1–40 nM) were on par with some
of the best known ligands for MGL, such as the Tn antigen. hMGL also
bound the natural beta-amyloid peptide containing a GalNAc-Tyr epitope.
STD NMR experiments provided structural insights into the molecular
basis of recognition. Finally, GalNAc-Tyr was selectively captured
by mMGL-2 positive dendritic cells. These results provide the first
evidence that GalNAc-Tyr modified proteins and/or peptides may be
ligands for hMGL and mMGL-2 and offer unique structures for the design
of MGL targeting agents
Dependence of inhibition on carbohydrate antigen structure.
<p>Measured IgG signals to Forssman disaccharide and tetrasaccharide (A) and four blood group A antigens (B) in the absence of added IgM (0 μg/mL) or in the presence of varying amounts of IgM (50–400 μg/mL).</p
Changes in serum IgG and IgM signals in a pooled serum sample in the presence of varying amounts of added IgG, IgA, or IgM.
<p>(A) Changes in IgG signals upon addition of IgM. (B) Changes in IgG signals upon addition of IgA. (C) Changes in IgM signals upon addition of IgG. (D) Changes in IgM signals upon addition of IgA. IgG and IgM signals in a pooled serum sample were first profiled on the array alone. Separately, the serum sample was pre-mixed with varying amounts of IgG, IgA, or IgM and then profiled on the array. The box plots depict the range of IgG or IgM signals (on a log base 2 scale) alone or in the presence of varying amounts of IgG, IgA, or IgM. The line in the middle of the box is the median signal, the box spans 1 standard deviation above and below the median, and the whiskers represent 2 standard deviations above or below the median. The only significant decreases observed are for IgG signals upon addition of IgM.</p
Competition between serum IgG, IgA, and IgM anti-glycan antibodies.
<p>(A) Addition of IgM and IgA to IgG. Polyclonal IgG isolated from serum was first profiled on the array alone. Separately, IgG was premixed with varying amounts of IgM or IgA and then profiled on the array. For each array component, the change in IgG signal in the presence of IgM or IgA was determined. The box plots depict the range of IgG changes (on a log base 2 scale) measured on the array upon addition of 4 serum equivalents of IgM or IgA. The line in the middle of the box is the median, the box spans 1 standard deviation above and below the median, and the whiskers represent 2 standard deviations above or below the median. (B) Addition of IgG and IgA to IgM. An analogous protocol as above was used to evaluate effects of IgG and IgA on IgM signals. (C) Addition of IgM and IgG to IgA. An analogous protocol as above was used to evaluate effects of IgG and IgM on IgA signals. The box plots demonstrate significant decreases in IgG and IgA signals in the presence of IgM for the vast majority of array components.</p
High-Throughput Profiling of Anti-Glycan Humoral Responses to SIV Vaccination and Challenge
<div><p>Recent progress toward an HIV vaccine highlights both the potential of vaccines to end the AIDS pandemic and the need to boost efficacy by incorporating additional vaccine strategies. Although many aspects of the immune response can contribute to vaccine efficacy, the key factors have not been defined fully yet. A particular area that may yield new insights is anti-glycan immune responses, such as those against the glycan shield that HIV uses to evade the immune system. In this study, we used glycan microarray technology to evaluate anti-glycan antibody responses induced by SIV vaccination and infection in a non-human primate model of HIV infection. This comprehensive profiling of circulating anti-glycan antibodies found changes in anti-glycan antibody levels after both vaccination with the Ad5hr-SIV vaccine and SIV infection. Notably, SIV infection produced generalized declines in anti-glycan IgM antibodies in a number of animals. Additionally, some infected animals generated antibodies to the Tn antigen, which is a cryptic tumor-associated antigen exposed by premature termination of <i>O</i>-linked glycans; however, the Ad5hr-SIV vaccine did not induce anti-Tn IgG antibodies. Overall, this study demonstrates the potential contributions that glycan microarrays can make for HIV vaccine development.</p></div
Comparison of anti-glycan antibody profiles in macaques and humans.
<p>Heat map showing glycan binding of circulating IgG (A) and IgM (B) in pre-vaccinated macaques (n = 38) and healthy humans (n = 30). Columns correspond to individual glycans organized into glycan families (see legend). Rows represent individual humans and macaques, which have been sorted by hierarchical clustering. Macaques and humans have highly similar repertoires of anti-glycan antibodies. Highlighted glycans are (1) 2′FucLac, (2) Hya8, (3) P1, (4) Ovalbumin, (5) LeA, (6) alpha-gal, (7) Forssman di, (8) Ac-Tn(Thr)-G-21, and (9) Ac-Tn(Thr)-Tn(Thr)-Tn(Thr)-G. Normalized data are plotted on a log 2 scale with a floor value of 7.2 (colored black).</p
Post-vaccination Tn responses.
<p>Five of the 38 macaques showed increases in anti-Tn IgG (A) and/or IgM (B). For these 5 animals, blue bars indicate the average fold change for 23 low density Tn glycoproteins occurring within 8 weeks of SIV infection (overall week 46–week 38). Red bars show the fold change after 26 weeks of infection (overall week 64–week 38). The animals are ordered in increasing levels of viremia.</p
Post-vaccination changes in anti-glycan antibodies.
<p>Heat map showing changes in IgG (A) and IgM (B) circulating anti-glycan antibodies that occurred after vaccination (wk 38–wk 0). Rows correspond to individual macaques (n = 38) sorted by vaccine groups described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075302#pone-0075302-t001" target="_blank">Table 1</a>. Columns correspond to glycans grouped according to glycan family. Red indicates increases, and decreases are shown in blue. Non-significant changes (<4×) are white. As indicated by the arrows, Manα1-6Manβ had the most number of antibody changes, OSM had the largest increase in antibody levels, and LeY had the largest decrease. The dashed horizontal line marks the macaque with the large decline in anti-LeY IgM levels, and the solid line indicates the macaque with the largest OSM increase.</p