Mammalian O-mannosylation of cadherins and plexins is independent of protein O-mannosyltransferases 1 and 2

Abstract

Protein O-mannosylation is found in yeast and metazoans, and a family of conserved orthologous protein O-mannosyltransferases is believed to initiate this important post-translational modification. We recently discovered that the cadherin superfamily carries O-linked mannose (O-Man) glycans at highly conserved residues in specific extracellular cadherin domains, and it was suggested that the function of E-cadherin was dependent on the O-Man glycans. Deficiencies in enzymes catalyzing O-Man biosynthesis, including the two human protein O-mannosyltransferases, POMT1 and POMT2, underlie a subgroup of congenital muscular dystrophies designated ␣-dystroglycanopathies, because deficient O-Man glycosylation of ␣-dystroglycan disrupts laminin interaction with ␣-dystroglycan and the extracellular matrix. To explore the functions of O-Man glycans on cadherins and protocadherins, we used a combinatorial gene-editing strategy in multiple cell lines to evaluate the role of the two POMTs initiating O-Man glycosylation and the major enzyme elongating O-Man glycans, the protein O-mannose ␤-1,2-N-acetylglucosaminyltransferase, POMGnT1. Surprisingly, O-mannosylation of cadherins and protocadherins does not require POMT1 and/or POMT2 in contrast to ␣-dystroglycan, and moreover, the O-Man glycans on cadherins are not elongated. Thus, the classical and evolutionarily conserved POMT O-mannosylation pathway is essentially dedicated to ␣-dystroglycan and a few other proteins, whereas a novel O-mannosylation process in mammalian cells is predicted to serve the large cadherin superfamily and other proteins.Protein O-glycosylation of the O-mannose type was originally thought to be found only in yeast and fungi, but studies over the last 30 years have identified O-Man 2 glycans and specific glycoproteins carrying O-Man glycans in human and rodents (1-9). The basement membrane glycoprotein ␣-dystroglycan (␣-DG) was for some time the only well characterized O-mannosylated protein known in mammals despite evidence that O-Man glycans constitute a major part of the total O-glycans in the brain (1,2,8,9). O-Mannosylation of ␣-DG is essential for assembly and function of the dystrophin-glycoprotein complex that links the cytoskeleton with the extracellular matrix, and deficiencies in all of the enzymes involved in the O-Man glycosylation underlie a subgroup of congenital muscular dystrophies (10 -12). More recently, the human O-Man glycoproteome was characterized, and it was found that the large superfamily of cadherins (cdhs) and protocadherins (pcdhs) are also decorated with ␣-linked O-Man glycans on extracellular cadherin (EC) domains. The attachment sites of these O-Man glycans appear to be highly conserved throughout evolution (13,14); moreover, O-mannosylation of E-cadherin was suggested to be crucial for E-cadherin-mediated cell adhesion (15,16).O-Man glycosylation in metazoans is initiated in the endoplasmic reticulum (ER) by transfer of mannose from dolichol monophosphate-activated mannose to serine and threonine by the POMT1 and POMT2 p...

Global aspects of viral glycosylation

“…Most types of O-glycosylation are carried out in the secretory pathway, but cytosolic and nuclear proteins can also be glycosylated with N-acetylglucosamine (GlcNAc) by a cytosolic glycosyltransferase (Moremen et al 2012;Yang and Qian 2017). Some types of O-glycosylation are specific to distinct classes of proteins or domains (Moremen et al 2012;Haltom and Jafar-Nejad 2015;Larsen et al 2017). In this review the term O-glycosylation will refer to GalNAc-or mucin-type O-glycosylation, which is one of the most widespread forms of protein O-glycosylation (Bennett et al 2012).…”

In vitro analysis of arrhythmogenic cardiomyopathy associated desmoglein-2 (DSG2) mutations reveals diverse glycosylation patterns

“…The recently described molecular structure of the extracellular domains of DSG2, revealed three O-mannosylation sites (p.T480, p.T482 and p.T484) in the fourth extracellular domain of DSG2 [16]. Omannosylations are rare sugar modifications, which have been recently identified in several members of the cadherin family [32][33][34]. In our study, no carbohydrates were detectable after enzymatic digestion using PNGase F suggesting that O-mannosylation was not present or below the limit of detection of Pro-Q® Emerald 300 glycoprotein staining, respectively.…”

ER transmembrane protein TMTC3 contributes to O-mannosylation of E-cadherin, Cellular Adherence and Embryonic Gastrulation

“…TMTC1-4 were discovered to contribute to the O-mannosylation proteome when a subset of substrates remained O-mannosylated in cells that lacked the known Omannosyltransferases, POMT1 and POMT2 (Larsen et al, 2017a(Larsen et al, , 2017b. A significant difference in O-mannosylation of the cadherin family of proteins was observed upon knockout of all four TMTCs in HEK293 cells (HEK293 SC/TMTC1,2,3,4 ) (Larsen et al, 2017a).…”

“…A new putative family of O-mannosyltransferases was recently discovered in mammals (Larsen et al, 2017a(Larsen et al, , 2017b. This family is comprised of four tetratricopeptide repeat (TPR)-containing proteins that appear to be ER polytopic transmembrane proteins called TMTC1-4 (transmembrane and TPR-containing proteins 1-4) (Della-Morte et al, 2011;Racapé et al, 2011;Cao et al, 2012;Sunryd et al, 2014;Larsen et al, 2017a;Li et al, 2018).…”