title: The Thr715Pro variant impairs terminal glycosylation of P-selectin.
abstract: P-selectin variant 715Pro is associated with lower concentrations of plasma P-selectin and reduced risk for thrombosis. We examined the influence of 715Pro on P-selectin synthesis, post-translational processing, surface expression and function using HEK293 cells, which do not express endogenous P-selectin. Mass spectrometry revealed that HEK293 cells produced recombinant P-selectin which has a glycosylation pattern comparable to platelet P-selectin. Compared to wild-type transfectants, 715Pro transfectants have ~50% less terminally glycosylated P-selectin and accumulate more immature P-selectin in Golgi. Following Brefeldin A treatment, the majority of 715Pro P-selectin is not modified by Golgi enzymes, while wild-type P-selectin undergoes complete modification. Flow cytometry revealed that 715Pro transfectants have ~20% less P-selectin on the cell surface compared to wild-type transfectants. Secretion of P-selectin by 715Pro transfectants was about 38% lower compared to wild-type transfectants. Binding of HL-60 cells to 715Pro transfectants was ~29% lower than to wild-type transfectants. This observation was confirmed by the presence of fewer platelet-monocyte aggregates (PMA) in the blood of healthy individuals and patients with angiographically proven atherosclerosis, carrying 715Pro P-selectin compared to individuals with wild-type P-selectin. We conclude that the 715Pro variant impairs terminal glycosylation of P-selectin in Golgi, leading to reduced amounts of mature P-selectin and subsequently less surface expression and secretion of P-selectin. The reduced surface expression of 715Pro P-selectin contributes to inefficient adhesion to HL-60 cells or monocytes.
authors.affiliation: Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
authors.forename: Hariharan
authors.initials: H
authors.lastname: Subramanian
authors.forename: Stepan
authors.initials: S
authors.lastname: Gambaryan
authors.forename: Simon
authors.initials: S
authors.lastname: Panzer
authors.forename: Thomas
authors.initials: T
authors.lastname: Gremmel
authors.forename: Ulrich
authors.initials: U
authors.lastname: Walter
authors.forename: Christine
authors.initials: C
authors.lastname: Mannhalter
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001792:Blood Platelets / Q000502:physiology; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D014644:Genetic Variation; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D057809:HEK293 Cells; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009000:Monocytes / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D019007:P-Selectin / Q000097:blood / Q000737:chemistry* / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D019007:P-Selectin; D011994:Recombinant Proteins