Haltiwanger, R. S. | Search bioRxiv

bioRxiv2022

Ultra-sensitive platelet proteome maps the O-glycosylation landscape and charts the response to thrombin dosage

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets tendency to pre-activate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1,300 proteins. Unbiased scanning for post-translational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterised sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal EMI domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new consensus sequence. Our data suggest that Protein O-fucosyltransferase 1 (POFUT1) is responsible for this modification. Secretion of MMRN1 was reduced in cells lacking POFUT1, supporting a key role of O-fucosylation in MMRN1 function. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3,800 proteins, which confirmed the platelet origin of releasate proteins by anti-correlation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The comprehensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis. Key PointsO_LIHigh-sensitivity proteome mapping of human platelets identifies O-glycosylation of platelet proteins at key functional sites C_LIO_LIPlatelet O-fucosyltransferase POFUT1 regulates the secretion of adhesive protein multimerin-1 (MMRN1) C_LI

biochemistry↗

bioRxiv2021

Metabolic heritage mapping: heterogenous pools of cytoplasmic nucleotide sugars are selectively utilized by various glycosyltransferases

Biosynthesis of macromolecules requires precursors such as sugars or amino acids, originating from exogenous/dietary sources, reutilization/salvage of degraded molecules or de novo synthesis. Since these sources are assumed to contribute to one homogenous pool, their individual contributions are often overlooked. Protein glycosylation uses monosaccharides from all the above sources to produce nucleotide sugars required to assemble hundreds of distinct glycans. Here we demonstrate that cells identify the origin/heritage of the monosaccharide, fucose, for glycosylation. We measured the contribution of GDP-fucose from each of these sources for glycan synthesis and found that different fucosyltransferases, individual glycoproteins, and linkage-specific fucose residues identify and select different GDP-fucose pools dependent on their heritage. This supports the hypothesis that GDP-fucose exists in multiple, distinct pools, not as a single homogenous pool. The selection is tightly regulated since the overall pool size remains constant. We present novel perspectives on monosaccharide metabolism, which may have general applicability.

biochemistry↗