Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing ␣-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I ␣-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded ␣1-antitrypsin variant (null (Hong Kong)) and of TCR␣. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded ␣1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent ␣1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of ␣1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has ␣1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.ER 3 quality control is an elaborate mechanism conserved from yeast to mammals, ensuring that newly synthesized proteins in the ER fold and assemble correctly and that only proteins that acquire their correct conformations are sorted further into the secretory pathway (1-4). During this process, proteins that fail to attain their native conformation due to mutations of the polypeptides or to ER stress conditions adverse for protein folding as well as orphan subunits are degraded in a process known as ER-associated degradation (ERAD) (3, 5-7). The recognition of misfolded proteins for ERAD is still poorly understood, but there is increasing evidence for a role of mannose trimming in the targeting of glycoproteins for ERAD (8, 9). In mammalian cells, overexpression of ER ␣-mannosidase I stimulates ERAD of misfolded glycoproteins (10, 11), whereas the ␣1,2-mannosidase inhibitors kifunensine and 1-deoxymannojirimycin stabilize misfolded glycoproteins (12-16). These observations suggested that Man 8 GlcNAc 2 isomer B, the major product of the ER ␣1,2-mannosidase, is a recognition marker for ERAD of glycoproteins, but this view is being challenged, since there is increasing evidence that trimming to smaller oligosaccharides occurs on ERAD substrates (10,(17)(18)(19). We previously cloned mouse EDEM (ER degradation enhancing ␣-mannosidase-like protein) as a cDNA whose expression is up-regulated by ER stress and showed that EDEM accelerates glycoprotein ERAD (20). EDEM is an integral ER membrane protein that has all the signature motifs of Class I ...

The adhesive interactions of cells with laminins are mediated by integrins and non-integrin-type receptors such as ␣-dystroglycan and syndecans. Laminins bind to these receptors at the C-terminal globular domain of their ␣ chains, but the regions recognized by these receptors have not been mapped precisely. In this study, we sought to locate the binding sites of laminin-10 (␣5␤1␥1) for ␣ 3 ␤ 1 and ␣ 6 ␤ 1 integrins and ␣-dystroglycan through the production of a series of recombinant laminin-10 proteins with deletions of the LG (laminin G-like) modules within the globular domain. We found that deletion of the LG4 -5 modules did not compromise the binding of laminin-10 to ␣ 3 ␤ 1 and ␣ 6 ␤ 1 integrins but completely abrogated its binding to ␣-dystroglycan. Further deletion up to the LG3 module resulted in loss of its binding to the integrins, underlining the importance of LG3 for integrin binding by laminin-10. When expressed individually as fusion proteins with glutathione S-transferase or the N-terminal 70-kDa region of fibronectin, only LG4 was capable of binding to ␣-dystroglycan, whereas neither LG3 nor any of the other LG modules retained the ability to bind to the integrins. Site-directed mutagenesis of the LG3 and LG4 modules indicated that Asp-3198 in the LG3 module is involved in the integrin binding by laminin-10, whereas multiple basic amino acid residues in the putative loop regions are involved synergistically in the ␣-dystroglycan binding by the LG4 module.

Lymphocyte homing to lymph nodes and Peyer's patches is mediated, in part, by adhesive interactions between L-selectin expressed by lymphocytes and L-selectin ligands displayed at the surface of the cuboidal endothelial cells lining the post-capillary venules within lymphoid aggregates. Candidate terminal oligosaccharide structures thought to be essential for effective L-selectin ligand activity include a sulfated derivative of the sialyl Lewis x tetrasaccharide. Cell type-specific synthesis of this oligosaccharide is presumed to require one or more alpha(1,3)fucosyltransferases, operating upon common 3'-sialylated and/or sulfated N-acetyllactosamine-type precursors. The identity of the alpha(1,3)fucosyltransferase(s) expressed in cells that bear L-selectin ligands has not been defined. We report here the molecular cloning and characterization of a murine alpha(1,3)fucosyltransferase locus whose expression pattern correlates with expression of high affinity ligands for L-selectin. In situ hybridization and immunohistochemical analyses demonstrate that this cDNA and its cognate alpha(1,3)fucosyltransferase are expressed in endothelial cells lining the high endothelial venules of peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. These expression patterns correlate precisely with the expression pattern of L-selectin ligands identified with a chimeric L-selectin/IgM immunohistochemical probe and by the high endothelial venule-reactive monoclonal antibody MECA-79. Transcripts corresponding to this cDNA are also detected in isolated bone marrow cells, a source rich in the surface-localized ligands for E- and P-selectins. Sequence and functional analyses indicate that this murine enzyme corresponds to the human Fuc-TVII locus. These observations suggest that Fuc-TVII participates in the generation of alpha(1,3)fucosylated ligands for L-selectin and provide further evidence for a role for this enzyme in E- and P-selectin ligand expression in leukocytes.