Human plasma protein N-glycosylation

Abstract

Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.

Keywords: Glycoproteins; Glycoproteomics; Immunoglobulins; N-glycosylation; Plasma; Serum.

Fig. 1

Schematic representation of the relative protein contribution to each specific glycan composition. To obtain these numbers, the contribution of a glycan composition to the total glycan pool of a given protein was multiplied by the abundance of that protein as well as the number of glycosylation sites confirmed to be occupied. Protein concentrations were taken from large studies when available and a mean value was calculated from the reported ranges otherwise. The molecular mass used is as reported by SDS-PAGE for the glycoproteins or calculated from the phenotype distribution for haptoglobin. The general Oxford notation was used for naming the glycan structures. For details on the calculation see Supplementary Table 1

Fig. 2

Typical reflectron positive mode MALDI-TOF-MS spectrum of the total N-glycosylation of pooled human plasma after enzymatic N-glycan release, ethyl esterification, and hydrophilic-interaction liquid chromatography (HILIC) enrichment [109]. Glycan species are assigned as [M+Na]⁺ on basis of the reviewed plasma structures. Where multiple options are possible, the most abundant has been used for assignment. Sialic acid orientation is on basis of observed mass after ethyl esterification, while the other linkages are presumed on basis of literature. For fucosylation, diantennary structures are reported to mostly carry an α1-6-linked fucose on the reducing end N-acetylglucosamine, while tri- and tetraantennary structures are reported to mostly have α1-3-linked antennary fucosylation in the form of Lewis X (or sialyl-Lewis X when the antenna carries an α2-3-linked sialic acid). For the tri- and tetraantennary structures, antennae representation has been simplified for readability purposes