Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain.

K Shailubhai,B.S Pukazhenthi,E.S Saxena,G.M Varma,I.K Vijay

doi:10.1016/s0021-9258(18)55341-2

K Shailubhai, B.S Pukazhenthi + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)55341-2

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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-linked oligosaccharide, soon after its transfer en bloc by the oligosaccharyl transferase complex in the lumen of endoplasmic reticulum.

Highlights

We have analyzed the functional domain structureoonef or more mannosyl residues may be removed and addirat mammary glucosidase I, an enzyme involved in N- tional sugars may be added as the glycoprotein reaches its linked glycoprotein processing, using biochemical andfinal destination, either within a membrane compartment of immunologicalapproaches.Theenzymecontains a the cell or secretion into the extracellular environment [1]
Such an orientation of thceatalytic domain may posal that the catalytic domain of the enzyme is oriented facilitate thesequentiapl rocessing of asparaginelinked oligosaccharide, soon after its transfer en bloc by the oligosaccharytrl ansferasecomplex in the lumen of endoplasmic reticulum
Characterization of Glucosidase I-the purified glucosidase I exhibited a band of 85 kDa on 10% SDS-PAGE under reducing conditions [21],it was resolved into two bands of 82 and 85 kDaon 12.5% SDS-PAGE in Tricinebuffer system [24] (Fig. la)

Summary

Introduction

We have analyzed the functional domain structureoonef or more mannosyl residues may be removed and addirat mammary glucosidase I, an enzyme involved in N- tional sugars may be added as the glycoprotein reaches its linked glycoprotein processing, using biochemical andfinal destination, either within a membrane compartment of immunologicalapproaches.Theenzymecontains a the cell or secretion into the extracellular environment [1]. A portion of the glucosidase I, following the digestion, wasused immediately for determination of the residual enzyme activity, and the remaining portion was analyzed by either 12.5% SDS-PAGE in Tricine buffer system [24] followedby silver staining or by immunoblotting. Inhibition by Treatment with NEM or Trypsin-For examining the inhibition of glucosidase I activity by NEM, intact or detergentpermeabilized membranes were preincubated with indicated concentrations of NEM for 10 min on ice, and the enzyme activity was determined.

Results

Conclusion