Proteogenomic Features of the Highly Polymorphic Histidine-rich Glycoprotein Arose Late in Evolution

Abstract

Histidine-rich glycoprotein (HRG) is a liver-produced protein circulating in human serum at high concentrations of around 125 μg/ml. HRG belongs to the family of type-3 cystatins and has been implicated in a plethora of biological processes, albeit that its precise function is still not well understood. Human HRG is a highly polymorphic protein, with at least five variants with minor allele frequencies of more than 10%, variable in populations from different parts of the world. Considering these five mutations we can theoretically expect 3⁵ = 243 possible genetic HRG variants in the population. Here, we purified HRG from serum of 44 individual donors and investigated by proteomics the occurrence of different allotypes, each being either homozygote or heterozygote for each of the five mutation sites. We observed that some mutational combinations in HRG were highly favored, while others were apparently missing, although they ought to be present based on the independent assembly of these five mutation sites. To further explore this behavior, we extracted data from the 1000 genome project (n ∼ 2500 genomes) and assessed the frequency of different HRG mutants in this larger dataset, observing a prevailing agreement with our proteomics data. From all the proteogenomic data we conclude that the five different mutation sites in HRG are not occurring independently, but several mutations at different sites are fully mutually exclusive, whereas others are highly intwined. Specific mutations do also affect HRG glycosylation. As the levels of HRG have been suggested as a protein biomarker in a variety of biological processes (e.g., aging, COVID-19 severity, severity of bacterial infections), we here conclude that the highly polymorphic nature of the protein needs to be considered in such proteomics evaluations, as these mutations may affect HRG's abundance, structure, posttranslational modifications, and function.

Keywords: 1000-genome project; histidine-rich glycoprotein (HRG); plasma proteomics; protein biomarker; proteogenomics; serum proteomics.

Graphical abstract

Fig. 1

Schematic structure of histidine-rich glycoprotein (HRG) and its gene variants with minor allele frequencies (MAFs) of more than 10%.A, schematic representation of protein domains of human serum HRG. HRG consists of six domains: two N-terminal domains (N1 and N2), two proline-rich regions (PRR1 and PRR2), a histidine-rich region (HRR), and a C-terminal domain (C). The disulfide bridges are depicted in dashed lines. B, partial structure and N-glycosylation sites on HRG. The structure of HRG is obtained by Swiss-model homology modeling based on Fetuin-B (6hpv.1.A) (3, 40, 41, 42, 43). The histidine-rich domain, which does not tend to crystallize, is represented with an oval. The residues of the N-glycosylation sites are shown as dark blue. A glycosylation site at Asn202 is induced by rs9898 (30). C, the frequencies of five abundant gene variants of HRG in different subpopulations. The subpopulations are named as follows: EUR, European; SAS, South Asian; AFR, African; AMR, American; EAS, East Asian. D, gene variants of HRG with MAF of more than 0.1 (i.e., 10% of the population) and their corresponding primate alleles (38).

Fig. 2

Gene variants of histidine-rich glycoprotein (HRG) measured by proteomics. Prototypical LC-MS traces of unique allele-specific peptides detected in serum HRG purified from different donors (Skyline was used for allele classification and quantification) of (A) the Arg448Cys substitution and (B) and the Asn493Ile substitution. The spectra from top to bottom are the LC-MS traces for donors being homozygote AA, heterozygote Aa/aA, or homozygote aa. Dark gray, Arg448; light gray, Cys448; dark orange, Ile493; light orange, Asn493. C, the frequencies of gene variants of HRG with minor allele frequencies of more than 10% as observed in the proteomics data from the 44 donors (left) and (for comparison) as extracted from the European genomics data from the 1000 genomes project (right).

Fig. 3

Pairwise co-occurrences of allele-specific mutations in histidine-rich glycoprotein between position 493 and other mutation sites.A, frequencies observed in the proteomics data (n = 32). B, theoretical frequencies of co-occurrences based on independent assembly. C, frequencies observed in the European subset from the 1000 genomes project.

Fig. 4

Theoretical (independent assembly) and experimental (1000 genome data) co-occurrences of all five allele frequencies in histidine-rich glycoprotein (HRG).A, occurrence percentage of the top 20 abundant combinations based on an independent assembly model (left) and the experimental 1000 genome data (right). The y-axis depicts the combinations of most frequent gene variants within HRG, with pairwise amino acids at position 180, 204, 340, 448, and 493. Cartoon figures with different colors represent these different allele combinations. Each figure represents 1% of the population. The pie chart insets depict the summed relative contribution of the 20 most frequently calculated/observed combinations. B, proposed evolutionary tree of the five mutations in HRG, enlightening why certain combinations of co-occurrences are unlikely to be observed. Each branch represents the most abundant allele combination of HRG s in different regions.