Meta-heterogeneity: Evaluating and Describing the Diversity in Glycosylation Between Sites on the Same Glycoprotein

Abstract

Mass spectrometry-based glycoproteomics has gone through some incredible developments over the last few years. Technological advances in glycopeptide enrichment, fragmentation methods, and data analysis workflows have enabled the transition of glycoproteomics from a niche application, mainly focused on the characterization of isolated glycoproteins, to a mature technology capable of profiling thousands of intact glycopeptides at once. In addition to numerous biological discoveries catalyzed by the technology, we are also observing an increase in studies focusing on global protein glycosylation and the relationship between multiple glycosylation sites on the same protein. It has become apparent that just describing protein glycosylation in terms of micro- and macro-heterogeneity, respectively, the variation and occupancy of glycans at a given site, is not sufficient to describe the observed interactions between sites. In this perspective we propose a new term, meta-heterogeneity, to describe a higher level of glycan regulation: the variation in glycosylation across multiple sites of a given protein. We provide literature examples of extensive meta-heterogeneity on relevant proteins such as antibodies, erythropoietin, myeloperoxidase, and a number of serum and plasma proteins. Furthermore, we postulate on the possible biological reasons and causes behind the intriguing meta-heterogeneity observed in glycoproteins.

Keywords: Acute phase proteins; Glycan; Glycoproteoforms; Glycosylation; Immunoglobulins; Macro-heterogeneity; Meta-heterogeneity; Micro-heterogeneity; Plasma/serum glycoproteins; Proteoforms.

Graphical abstract

Fig. 1

Overview of the glycosylation features discussed in this review that contribute to meta-heterogeneity. Glycan compositions were chosen to represent a small and a large variant within the given group (e.g., high-mannose). Tn, Core 1, and Core 2 O-glycosylation represent only a selection of the large group of possible O-glycan core structures, as the compositions themselves can be much larger. O-Acetylation has been positioned at a triantennary glycan but may in principle occur at any sialic acid (N-acetylneuraminic acid or N-glycolylneuraminic acid).

Fig. 2

Defining meta-heterogeneity. Although micro-heterogeneity (top left) describes the variation in glycan species at a given glycosylation site and macro-heterogeneity (bottom left) describes to which degree a glycosylation site is occupied, these terms are not sufficient to describe a higher order of regulation across a glycoprotein with multiple glycosylation sites. As such, we propose the concept of meta-heterogeneity (right) to describe the variation in glycosylation across all glycosylation sites of a given protein. In the chosen example (myeloperoxidase monomer; 1CXP) (90, 91) it can be seen that the N-glycosylation sites (yellow residues) are decorated by very different glycans, ranging from paucimannose to hybrid-/complex-type. This difference makes for a protein with high glycan meta-heterogeneity, indicating selective regulation and highlighting potential differences in structure–function relationships.

Fig. 3

Meta-heterogeneity in erythropoietin glycosylation. Mass spectrometric analysis indicated that Asn83 (top left) and Asn38 (bottom left) predominantly carried tetra-antennary glycans and could contain O-acetylation, while the most abundant glycan at Asn24 (bottom right) was di-antennary for which no O-acetylation was found. Based on the structure there is no apparent steric hindrance that could explain the lower complexity of Asn24 glycosylation, suggesting a site-specific layer of regulation. The structure was modified from the PDB entry 1BUY (with Lys to Asn substitution), and all glycoprotein structures (throughout the review) were generated by means of GLYCAM (www.glycam.org) (112, 113). Note that the glycans are highly dynamic and will likely occupy a large region around the shown structure. The mass spectrometric data were reused with permission from Yang et al. (111).

Fig. 4

Meta-heterogeneity cross talk found between properdin (complement factor P) N-glycosylation and C-mannosylation. As seen from the deconvoluted native mass spectra of properdin (top), diantennary N-glycoforms (A and B) co-occur with 11 to 15 C-mannosylations, while triantennary N-glycoforms (C–E) only co-occur with 15 C-mannosylations. In the properdin structure (bottom) (6RUS) (123) it can be seen that part of the C-mannosylation occurs in the same region as the N-glycan, making cross talk conceivable. One explanation may be that full C-mannose occupancy hinders the N-glycan from entering the protective pocket, thereby exposing the sugar for more extended modification by glycosyltransferases. Th mass spectrometric data were reused with permission from Bern et al. (122).

Fig. 5

Similarities and differences in glycan meta-heterogeneity between human and mouse myeloperoxidase (MPO). Human MPO (top left) is a highly meta-heterogenous glycoprotein with phosphomannosylation at Asn323, high-mannose species at Asn355 and Asn391, complex species at Asn483, and paucimannosylation/macro-heterogeneity at Asn729. Mouse MPO (top right) shows distinct differences in this meta-heterogeneity, for example, by carrying phosphomannose species at Asn329, Asn365, as well as Asn711. The structure of dimeric MPO (bottom) was generated from the human database (1CXP) (91) and shows representative glycosylation. The mass spectrometric data were reused with permission from Reiding et al. (90).