Title :
Glucosidase I , a transmembrane endoplasmic reticular
glycoprotein with a luminal catalytic
domain
Abstract :
- We have analyzed the functional domain structure of rat mammary glucosidase I , an enzyme involved in N-linked glycoprotein processing, using biochemical and immunological approaches
- The enzyme contains a high mannose type sugar chain that can be cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity
- Based on trypsin digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains : a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain
- A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes with trypsin
- This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific affinity gel
- Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme , consistent with its location as an integral membrane protein , whereas the 39-kDa fragment partitioned in the aqueous phase, a characteristic of soluble polypeptide
- These results indicate that glucosidase I is a transmembrane protein with a luminally oriented catalytic domain
- Such an orientation of the catalytic domain may facilitate the sequential processing of asparagine asparagine-linked oligosaccharide , soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum