A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin

Abstract

<jats:p>The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1β units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)<jats:sub>2</jats:sub>(α2)<jats:sub>n-2</jats:sub>(β)<jats:sub>n</jats:sub>oligomers (<jats:italic>n</jats:italic>&gt; 1) while Hp 2-2 occurs in cyclic (α2)<jats:sub>n</jats:sub>(β)<jats:sub>n</jats:sub>oligomers (<jats:italic>n</jats:italic>&gt; 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp’s antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp’s capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor’s genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.</jats:p>

Glycoproteoform Profiles of Individual Patients’ Plasma Alpha-1-Antichymotrypsin are Unique and Extensively Remodeled Following a Septic Episode

“…Similar to the observations made by Wu et al. on acid glycoprotein ( 40 ) and Tamara et al ( 27 ) on haptoglobin, the highly heterogeneous and complex AACT glycosylation profile precluded us from making a full spectral annotation. This high complexity is primarily caused by the high frequency and abundance of sialic acid ( N -acetylneuraminic acid) moieties.…”

“…They do not only expand the proteoform profiles immensely but also hamper the annotation of the glycan structures, as mass shifts caused by two fucosylation moieties or one sialylation moiety are hard to disentangle due to their alike masses (i.e., 291.1 and 292.1 Da, respectively). Hence, in line with the previous studies ( 27 , 40 ), we first treated the AACT samples with sialidase to remove all sialic acid moieties prior to MS analysis, which indeed simplified the resulting native mass spectra ( Supplementary Figures 3A, B ). Sialidase treatment proved to be very reproducible ( Supplementary Figure 3C ).…”

“…We and others have shown that high-resolution native mass spectrometry profiles of intact glycoproteins can provide valuable information about post-translational modifications (PTMs), but also another structural variability, such as genetic variants or mutations ( 26 , 27 , 40 , 57 , 58 ). Here we used a similar approach involving a rapid purification of AACT from plasma to investigate its proteoform profiles.…”

“…Moreover, plasma protein glycosylation is regulated by various factors such as the genetic background of the donor and their environment, making each patient likely unique ( 24 , 25 ). We recently reported evidence for this uniqueness, reporting that the proteoform profiles of two abundant plasma APPs, fetuin and haptoglobin, exhibit patient-specific attributes, some of them caused by genetic polymorphisms ( 26 , 27 ). These observations underline the urgency to investigate plasma proteins in patients individually, i.e.…”

Ribosome association primes the stringent factor Rel for tRNA-dependent locking in the A-site and activation of (p)ppGpp synthesis

“…All proteins were expressed, purified and characterized using SUMO-tagging strategy described earlier for E. coli RelA ( 33 ) and B. subtilis Rel ( 12 ) ( Supplementary Figure S1A-G ). The monomeric nature of 50 nM B. subtilis Rel and 100 nM E. coli RelA was confirmed by mass photometry ( 34 ) using Refeyn OneMP instrument (Refeyn Ltd.) ( Supplementary Figure S1H and I ).…”