Site-specific N-glycosylation analysis of soluble Fc\u03b3 receptor IIIb in human serum

Hirokazu Yagi,Nana Kawasaki,Lubka T Roumenina,Daisuke Takakura,Koichi Kato,Catherine Sautès-Fridman,Wolf Herman Fridman

doi:10.1038/s41598-018-21145-y

Abstract

Fc-receptors for immunoglobulin G (FcγRs) mediate a variety of effector and regulatory mechanisms in the immune system. N-glycosylation of FcγRs critically affects their functions which is well exemplified by antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis mediated by homologous FcγRIIIa and FcγRIIIb, respectively. Although several reports describe N-glycosylation profiles of recombinant FcγRIII glycoproteins, much remains unknown regarding their native glycoforms. Here we performed site-specific N-glycosylation profiling of a soluble form of FcγRIIIb purified from human serum based on mass spectrometric analysis. Our data indicate a distinct and common tendency of the glycoforms exhibited at each N-glycosylation site between the native and the previously reported recombinant FcγRIII glycoproteins. Among the six N-glycosylation sites of serum soluble FcγRIIIb, Asn45 was shown to be exclusively occupied by high-mannose-type oligosaccharides, whereas the remaining sites were solely modified by the complex-type oligosaccharides with sialic acid and fucose residues. The results of our endogenous FcγRIII glycoform analyses are important for the optimization of therapeutic antibody efficacy.

Highlights

Various effector and regulatory mechanisms in the immune system are mediated through the interactions between immunoglobulins (Igs) and their cognate receptors that recognize their Fc portions[1,2,3]
As previously reported[9], sFcγRIIIb was purified from a pool of human serum by a series of chromatographic procedures
Consistent with the previous report[9], we obtained a smear, instead of a band, of the sFcγRIIIb, which was stained with Coomassie Brilliant Blue (CBB), indicating that the serum sFcγRIIIb is highly glycosylated with considerable heterogeneity (Supplementary Fig. 1)

Summary

Results and Discussion

As previously reported[9], sFcγRIIIb was purified from a pool of human serum by a series of chromatographic procedures. We analyzed the purified serum sFcγRIIIb using LC-MS/MS after the GluC and chymotrypsin digestions, and identified and semiquantified (i) 14 glycoforms on Asn[38], (ii) 6 glycoforms on Asn[45], (iii) 30 glycoforms on Asn[64], (iv) 45 glycoforms on Asn[74], (v) 55 glycoforms on Asn[162], and (vi) 15 glycoforms on Asn[169] (Fig. 2, Table 1, and Supplementary Tables 1–6). Our data revealed that each N-glycosylation site of the serum sFcγRIIIb was modified in a distinct fashion in terms of number, composition, and variability of N-glycans. Our previous N-glycosylation profiling of the recombinant sFcγRIIIb expressed by baby hamster kidney (BHK) cells identified highly-branched sialyl N-glycans but not the non-reducing terminal fucose residues[27]. Peptide Sequence (Y)SPEDNSTQW(F) (W)FHNESLI(S) (F)IDAATVNDSGEY(R) (Y)RCQTNL(S) (D)SGSYFCRGLVGSKNVSSE(T) (E)TVNITITQGLA(V)

Enzyme Chymotrypsin Chymotrypsin Chymotrypsin Chymotrypsin GluC GluC

Methods

Additional Information