Introduction
(Abstract)
Oligosaccharide structures are commonly found covalently linked to Asn residues on cellular and secreted proteins (1, 2, 3). The biosynthesis and maturation of these structures occurs within the membranes of the endoplasmic reticulum (ER) and the Golgi complex. A large, branched oligosaccharide structure is initially synthesized as a lipid-anchored precursor that is subsequently transferred to form an amide linkage to Asn side chains of newly synthesized polypeptides as they are co-translationally translocated through the ER membrane. Once transferred to protein, the precursor structure is immediately trimmed by removal of glucose and mannose residues within the compartment space of the ER and the Golgi complex. Following the trimming stages, the carbohydrate structures are built back up by the addition of a distinctive set of sugars to form an array of complex type structures that are found on cell surface and secreted glycoproteins. The early oligosaccharide trimming steps in the ER have two major roles: (a) they produce trimming intermediates that are selectively recognized by chaperonins within the compartment space of the ER that help facilitate protein folding and (b) they act as a timing step to target proteins that have not folded correctly for degradation (2). One of the key enzymes that acts at the branch point between glycoprotein biosynthesis and degradation is an enzyme termed ER a-mannosidase I (also known as ER Class 1 a1,2-mannosidase). The enzyme acts as the first step in mannose trimming for glycoproteins that are destined to be transport through the secretory pathway. In addition, the enzyme appears to acts as the rate limiting timing step for targeting unfolded glycoproteins for degradation (4). Thus, mutant proteins synthesized and extruded into the lumen of the ER, including the gene products in many human genetic diseases, are selectively targeted for degradation by the action of ER a-mannosiadase I. The enzyme is conserved from yeast to mammals and the structure of the yeast and human forms have been determined (2, 5, 6). Based on the determination of the protein structure in the presence and absence of small inhibitory substrate mimics, the enzyme has been hypothesized to have an unusual catalytic mechanism directly employing Ca+2 in substrate binding and possibly in catalysis. The enzyme is highly specific for the recognition of its substrate, cleaving only one of four potential a1,2-mannose residues from the oligosaccharide processing intermediate. Despite the rigid substrate specificity of the ER enzyme, a single amino acid substitution has been shown to confer a broader specificity to the enzyme resulting in a conversion to a broader specificity enzyme that is able to cleave all four a1,2-mannosyl residues, similar to homologous enzymes in the Golgi complex (7).