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Characterization of Endogenous Human Fc?RIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

ABSTRACT: The importance of IgG glycosylation, Fc-gamma receptor (Fc?R) single nucleotide polymorphisms and Fc?R copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of Fc?Rs in modulating the Fc?R-IgG interaction based on the association between the glycosylation of recombinant Fc?Rs and the kinetics and affinity of the Fc?R-IgG interaction. Although glycosylation of recombinant Fc?Rs has been recently characterized, limited knowledge exists on the glycosylation of endogenous human Fc?Rs. In order to improve the structural understanding of Fc?Rs expressed on human cells we characterized the site specific glycosylation of native human Fc?RIII from neutrophils of 50 healthy donors and from matched plasma for 43 of these individuals. Through this analysis we have confirmed site specific glycosylation patterns previously reported for soluble Fc?RIII from a single donor, identified Fc?RIIIb specific Asn45 glycosylation and an allelic effect on glycosylation at Asn162 of Fc?RIIIb. Identification of Fc?RIIIb specific glycosylation allows for assignment of Fc?RIIIb alleles and relative copy number of the two alleles where DNA/RNA is not available. Intriguingly the types of structures found to be elevated at Asn162 in the NA2 allele have been shown to destabilize the Fc:Fc?RIII interaction resulting in a faster dissociation rate. These differences in glycosylation may in part explain the differential activity reported for the two alleles which have similar in vitro affinity for IgG.

SUBMITTER: Washburn N

PROVIDER: S-EPMC6398215 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

Washburn Nathaniel N Meccariello Robin R Duffner Jay J Getchell Kristen K Holte Kimberly K Prod'homme Thomas T Srinivasan Karunya K Prenovitz Robert R Lansing Jonathan J Capila Ishan I Kaundinya Ganesh G Manning Anthony M AM Bosques Carlos J CJ

Molecular & cellular proteomics : MCP 20181217 3

The importance of IgG glycosylation, Fc-gamma receptor (FcγR) single nucleotide polymorphisms and FcγR copy number variations in fine tuning the immune response has been well established. There is a growing appreciation of the importance of glycosylation of FcγRs in modulating the FcγR-IgG interaction based on the association between the glycosylation of recombinant FcγRs and the kinetics and affinity of the FcγR-IgG interaction. Although glycosylation of recombinant FcγRs has been recently char ...[more]

PMID: 30559323

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Project description:We demonstrate a new approach for the site-specific identification and characterization of protein N-glycosylation. It is based on a nano-liquid chromatography microarray-matrix assisted laser desorption/ionization-MS platform, which employs droplet microfluidics for on-plate nanoliter reactions. A chromatographic separation of a proteolytic digest is deposited at a high frequency on the microarray. In this way, a short separation run is archived into thousands of nanoliter reaction cavities, and chromatographic peaks are spread over multiple array spots. After fractionation, each other spot is treated with PNGaseF to generate two correlated traces within one run, one with treated spots where glycans are enzymatically released from the peptides, and one containing the intact glycopeptides. Mining for distinct glycosites is performed by searching for the predicted deglycosylated peptides in the treated trace. An identified peptide then leads directly to the position of the "intact" glycopeptide clusters, which are located in the adjacent spots. Furthermore, the deglycosylated peptide can be sequenced efficiently in a simple collision-induced dissociation-MS experiment. We applied the microarray approach to a detailed site-specific glycosylation analysis of human serum IgM. By scanning the treated spots with low-resolution matrix assisted laser desorption/ionization-time-of-flight-MS, we observed all five deglycosylated peptides, including the one originating from the secretory chain. A detailed glycopeptide characterization was then accomplished on the adjacent, untreated spots with high mass resolution and high mass accuracy using a matrix assisted laser desorption ionization-Fourier transform-MS. We present the first detailed and comprehensive mass spectrometric analysis on the glycopeptide level for human polyclonal IgM with high mass accuracy. Besides complex type glycans on Asn 395, 332, 171, and on the J chain, we observed oligomannosidic glycans on Asn 563, Asn 402 and minor amounts of oligomannosidic glycans on the glycosite Asn 171. Furthermore, hybrid type glycans were found on Asn 402, Asn 171 and in traces Asn 332.

Dataset Information

Characterization of Endogenous Human Fc?RIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

Publications

Characterization of Endogenous Human FcγRIII by Mass Spectrometry Reveals Site, Allele and Sequence Specific Glycosylation.

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