Role of N-glycosylation of the SEA module of rodent Muc3 in posttranslational processing of its carboxy-terminal domain

Abstract

A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A, and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two, or three N-glycans, but was partially inhibited when lacking four, five, and six N-glycans. In all, 2%, 48%, 85%, and 73% of the products from N3457A, N34567A, N134567A, and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.

Activity of recombinant cysteine-rich domain proteins derived from the membrane-bound MUC17/Muc3 family mucins

“…These data suggest that the full length CRD1-L-CRD2 protein with an intact SEA domain are required for the protein to be fully functional. He, et al have shown that within the SEA module several N-glycosylation sites flanking the GSVVV cleavage site may control the extent of cleavage that occurs at this cleavage site, indicating the functional importance of the full SEA module [22]. Further experiments with site directed mutagenesis of the SEA cleavage site would be indicated to determine more specifically if this plays a role in recombinant mucin function, and also what part of the SEA module is important for functionality.…”

EGF-Containing Membrane-Bound Mucins: A Hidden ErbB2 Targeting Pathway?

“…Residues other than those involved in the autoproteolysis were found to have an impact on the stabilization of the noncovalent subunit interaction 31 and on the level of N-glycosylation whose modifications disrupt mucin functions. 32 All TM mucins of interest exhibit the canonical SEA domain except MUC4. Nevertheless, as previously demonstrated, the DI located between EGF1 and EGF2 domains can be considered as a degenerate SEA-like domain.…”

“…However, inhibition of the cleavage potency of the SEA domain has abolished the whole function of the mucins regarding proliferation and migration. 32,33 There is also evidence of a second cleavage site in several mucins, 51,59 which would involve the release of a full soluble extracellular subunit and a free cytoplasmic subunit leading to transduction of the signal to the nucleus. 60 Another role for this SEA domain could be to form a ligand−receptor combination from a unique gene.…”

“…By interacting directly or indirectly with adhesion-associated proteins such as lectins, selectins, or integrins, they could regulate cell trafficking (cell–cell adhesion, cell–cell communication, and cell–matrix interaction). ,, Finally, they are also involved in the regulation of the cell cycle because they can induce proliferation, differentiation, or migration. ,, Among all of these functions, most are carried out by the glycans of the O-glycosylated subunit. Nevertheless, the EGF-containing mucins are involved in cell signaling and regulation processes, − in healthy conditions, from tissue regeneration, wound healing, and embryogenesis and fetus development. , …”

“…The autoproteolytic pattern is not necessarily conserved but sometimes is replaced by a specific protease recognition site, maintaining its ability to be cleaved. Residues other than those involved in the autoproteolysis were found to have an impact on the stabilization of the noncovalent subunit interaction and on the level of N-glycosylation whose modifications disrupt mucin functions …”