Initiation of Protein O Glycosylation by the Polypeptide GalNAcT-1 in Vascular Biology and Humoral Immunity

Abstract

Core-type protein O glycosylation is initiated by polypeptide N-acetylgalactosamine (GalNAc) transferase (ppGalNAcT) activity and produces the covalent linkage of serine and threonine residues of proteins. More than a dozen ppGalNAcTs operate within multicellular organisms, and they differ with respect to expression patterns and substrate selectivity. These distinctive features imply that each ppGalNAcT may differentially modulate regulatory processes in animal development, physiology, and perhaps disease. We found that ppGal NAcT-1 plays key roles in cell and glycoprotein selective functions that modulate the hematopoietic system. Loss of ppGalNAcT-1 activity in the mouse results in a bleeding disorder which tracks with reduced plasma levels of blood coagulation factors V, VII, VIII, IX, X, and XII. ppGalNAcT-1 further supports leukocyte trafficking and residency in normal homeostatic physiology as well as during inflammatory responses, in part by providing a scaffold for the synthesis of selectin ligands expressed by neutrophils and endothelial cells of peripheral lymph nodes. Animals lacking ppGalNAcT-1 are also markedly impaired in immunoglobulin G production, coincident with increased germinal center B-cell apoptosis and reduced levels of plasma B cells. These findings reveal that the initiation of protein O glycosylation by ppGal NAcT-1 provides a distinctive repertoire of advantageous functions that support vascular responses and humoral immunity.

Severe Impairment of Leukocyte Recruitment in ppGalNAcT-1–Deficient Mice

“…Other transferases might contribute to O-glycosylation as well. Consistent with our findings, flow chamber experiments showed that neutrophils from Galnt1 2/2 mice had a reduced, but not abolished, binding capacity to P-selectin and E-selectin under flow conditions (26).…”

“…P-selectin-dependent leukocyte rolling in vivo is significantly impaired in Galnt1 2/2 mice Leukocytes from Galnt1 2/2 mice have a reduced P-selectinbinding capacity in vitro (26). To investigate the functional consequences in vivo, leukocyte rolling was analyzed in 28 venules of five Galnt1 2/2 mice and compared with leukocyte rolling in 20 venules of four littermate controls.…”

“…We extended these findings by using different inflammation animal models, confirming the predominant role of ppGalNAcT-1 in attaching functionally relevant O-linked glycans to selectin ligands under inflammatory conditions. The loss of ppGalNAcT-1 had a severe impact on neutrophil recruitment in the thioglycollate-induced peritonitis model (26). To differentiate between hematopoietic and nonhematopoietic ppGalNAcT-1 in leukocyte recruitment, we generated bone marrow chimeras and investigated leukocyte recruitment into the peritoneal cavity after thioglycollate injection.…”

“…Recent work by Tenno et al (26) provides evidence that Oglycosylation by ppGalNAcT-1, by adding galactosamine to serine or threonine residues of a protein backbone, is crucial for binding P-selectin and E-selectin IgM in vitro (26). Furthermore, leukocyte rolling on E-selectin and P-selectin in autoperfused flow chamber assays was significantly reduced in Galnt1 2/2 mice.…”

“…Surprisingly, genetic elimination of ppGalNAcT-1 in the mouse did not abolish Oglycan formation in vivo (23,24), indicating at least partially redundant functions of the different ppGalNAcT isoforms (24,25). However, elimination of ppGalNAcT-1 results in a bleeding disorder caused by reduced plasma levels of different blood coagulation factors and impaired leukocyte recruitment and homing under normal and inflammatory conditions (26).…”