Genotype-dependent N-glycosylation and novel O-glycosylation affect the activation of histidine-rich glycoprotein by plasmin

Introduction

Histidine-rich glycoprotein (HRG) is an abundant plasma glycoprotein that regulates various biological processes, including coagulation and immune response. HRG has been identified as a biomarker for multiple diseases, such as COVID-19, AIDS, asthma, renal disease, pulmonary disease, and thrombotic disorders. In humans, HRG is highly polymorphic, with five variants having a maximum allele frequency (MAF) over 10%, affecting its structure and function. The Pro204Ser mutation, notably, introduces a putative N-glycosylation site at Asn202, which is associated with reduced pregnancy rates, aging, and mortality. Other N-glycosylation sites in HRG also impact functions such as cell proliferation and cancer regulation. Despite HRG's significant role in homeostasis, the precise effects of its mutations and glycosylation remain unclear.

Methods

We optimized an immobilized metal affinity chromatography (IMAC) purification protocol, leveraging HRG's histidine-rich region to efficiently enrich the protein from serum. We then used LC-MS/MS-based (glyco)proteomics to characterize HRG mutants and glycosylation in the sera of COVID-19 infected and healthy individuals, as well as in recombinant variants with mutations at positions 204 and 493. To further explore HRG mutations, we extracted data from the 1000 Genomes Project, assessing the frequency of these mutants. Additionally, we examined plasmin-induced cleavage of HRG to understand the functional and structural interplay between HRG function, mutations, and glycosylation.

Preliminary Data

Our (glyco)proteomics approach identified the genetic make-up of HRG in the sera of all donors, revealing their homozygotic or heterozygotic allotypes and the glycosylation profiles of each purified HRG sample. We found strong correlations between specific HRG allotype mutations, some of which were either mutually exclusive or supportive. These findings were partially corroborated by data from the 1000 Genomes Project.

We also measured the glycosylation profiles of endogenous plasma HRG and recombinantly produced HRG (in CHO and HEK293 cells), showing distinctive patterns. The Pro204Ser-specific Asn202 site, with a ~50% MAF frequency, had high occupancy by complex-type N-glycans. Additionally, we identified novel O-glycosylation sites at HRG Thr273 and Thr274, with over 90% occupancy across all HRG variants. As these O-glycosylation sites are near a proposed plasmin-cleavage site (Lys275), we assessed plasmin-induced cleavage kinetics and specificity. The assay showed that plasmin-induced cleavage was significantly influenced by sialylation, particularly of the O-linked glycans. Moreover, the Pro204Ser mutation notably impacted plasmin-induced cleavage. Our findings reveal the intricate interplay of glycoproteogenomic features within HRG, suggesting how these factors may contribute to HRG's regulatory role in homeostasis in vivo.