Purification and characterization of trimming glucosidase I from pig liver

Abstract

Trimming glucosidase I has been purified about 400‐fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N‐glycoprotein as shown by its binding to concanavalin A—Sepharose and its susceptibility to endo‐β‐N‐acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non‐specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N‐linked high‐mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal α1–2‐linked glucose residue from natural Glc3‐Man9‐GlcNAc2, but is inactive towards Glc2‐Man9‐GlcNAc2 or nitrophenyl‐/methyl‐umbelliferyl‐α‐glucosides. The enzyme displays a pH optimum close to 6.4. does not require metal ions for activity and is strongly inhibited by 1‐deoxynojirimycin (Ki∼ 2.1 μM), N,N‐dimethyl‐1‐deoxynojirimycin (Ki∼ 0.5 μM) and N‐(5‐carboxypentyl)‐1‐deoxynojirimycin (Ki∼ 0.45 μM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross‐react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross‐reactivity.

Cloning and Expression of Glucosidase I from Human Hippocampus

“…In this paper, we describe the cloning of glucosidase I from the human hippocampus and the characterization of the enzyme after expression in COS 1 cells. The molecular and enzymic properties of the expressed enzyme [mo-lecular mass, N-glycoprotein nature, substrate specificity and inhibition by 1 -deoxynojirimycin and N-alkyl derivatives of 1-deoxynojirimycin (dNM)] are consistent with data determined for the enzyme purified from pig liver [7]. Further similarities include an inferred type-I1 transmembrane topology of the glucosidase I protein as well as its ER location .…”

“…Glucosidase I purified from pig liver according to [7] was subjected to tryptic cleavage as described in Materials and Methods. The tryptic peptides were seperated by HPLC and their amino acid sequence determined by Edman degradation (Fig.…”

“…Tryptic cleavage of glucosidase I. 400 pg glucosidase I, purified from pig liver [7], was resuspended in 350 p1 0.5 M TrisMCl, pH 8.6, containing 5 M guanidine hydrochloride. Disulfide bonds were reduced with 8 pmol dithiothreitol, followed by carboxymethylation of sulfhydryl groups with 24 pmol iodoacetic acid.…”

β‐Glucosidase activity towards a bile acid glucoside in human liver

“…Enzyme activity at pH 5.0 was, however, less sensitive to inhibition by EDTA, with 50% inhibition occurring at an EDTA concentration of 10 mM. The metal ion requirement observed for fl-glucosidase activity toward chenodeoxycholie acid glucoside is in contrast to other a-or fl-glueosidase activities, which have not been described to be metal ion dependent or affected by metal ion chelating agents such as EDTA [5,8,16]. However, various g-mannosidases have been shown to require divalent metal ions for activity and are inhibited by EDTA [17][18][19].…”

Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics

“…Briefly, newly synthesized N-linked glycoproteins are glycosylated with 14 sugars donated from a dolichol-linked lipid via oligosaccharyl transferase (Das and Heath 1980). Following the sequential removal of the terminal glucose by glucosidase I (Hettkamp et al 1984;Bause et al 1989), the glycoprotein may then be recognized by the protein malectin (Schallus et al 2008;Chen et al 2011;Galli et al 2011), potentially for presentation to glucosidase II for removal of the second glucose residue (Burns and Touster 1982;Reitman et al 1982;Pelletier et al 2000). The resulting mono-glucosylated glycoprotein is then a client for association with calnexin or calreticulin (Hammond et al 1994;Zapun et al 1997;Schrag et al 2003).…”