Role of sulfhydryl groups in the function of glucosidase I from mammary gland

Department of Animal Sciences, University of Maryland, College Park 20742.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/1993; 268(9):6445-52.
Source: PubMed


Glucosidase I initiates the processing of asparagine-linked glycoproteins by excising the distal alpha 1,2-linked glucosyl residue from the Glc3Man9GlcNAc2 oligosaccharide, soon after its en bloc transfer from the lipid-linked donor to the nascent polypeptide. 1-Deoxynojirimycin, an analog of D-glucose, is a potent competitive inhibitor of the enzyme. Sulfhydryl-seeking reagents also strongly inhibit the enzyme, implying the involvement of an -SH group in its activity. To test this hypothesis, glucosidase I was purified from the rat mammary gland and its active site was loaded with 1-deoxynojirimycin, to protect such a group(s), while -SH groups on the remaining surface of the enzyme were blocked with N-ethylmaleimide or para-chloromercuriphenylsulfonic acid. Deoxynojirimycin was removed by dialysis to expose the active site -SH group(s). This group(s) was then tagged with 3-(N-maleimidopropionyl)biocytin (MPB) and detected with 125I-streptavidin on Western blots. A series of experiments is presented to show that indeed a critical -SH group(s) is located within the catalytic site of the enzyme. Additionally, the enzyme also possesses one or more sulfhydryls and disulfide bonds in its primary structure. The experimental approach outlined here should apply to identify reactive sulfhydryl groups in other catalytically active proteins.

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    • "α-Glucosidase I catalysis, like other glycosidases, is controlled by carboxylic acid residues (Koshland, 1953), as previously demonstrated by selective chemical modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Dhanawansa et al., 2002). Other residues, including Arg, Cys, and Trp, were reported to be likely participants in the binding site of mammalian α-glucosidase I based on chemical modification (Pukazhenthi et al., 1993; Romaniouk and Vijay, 1997). Also, mutated α-glucosidase I isolated from a patient with congenital disorder of glycosylation type IIb showed that together, Arg 486 Thr and Phe 652 Leu substitutions largely inactivated the enzyme (De Praeter et al., 2000; Volker et al., 2002). "
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    ABSTRACT: Alpha-glucosidase I initiates the trimming of newly assembled N-linked glycoproteins in the lumen of the endoplasmic reticulum (ER). Site-specific chemical modification of the soluble alpha-glucosidase I from yeast using diethylpyrocarbonate (DEPC) and tetranitromethane (TNM) revealed that histidine and tyrosine are involved in the catalytic activity of the enzyme, as these residues could be protected from modification using the inhibitor deoxynojirimycin. Deoxynojirimycin could not prevent inactivation of enzyme treated with N-bromosuccinimide (NBS) used to modify tryptophan residues. Therefore, the binding mechanism of yeast enzyme contains different amino acid residues compared to its mammalian counterpart. Catalytically active polypeptides were isolated from endogenous proteolysis and controlled trypsin hydrolysis of the enzyme. A 37-kDa nonglycosylated polypeptide was isolated as the smallest active fragment from both digests, using affinity chromatography with inhibitor-based resins (N-methyl-N-59-carboxypentyl- and N-59-carboxypentyl-deoxynojirimycin). N-terminal sequencing confirmed that the catalytic domain of the enzyme is located at the C-terminus. The hydrolysis sites were between Arg(521) and Thr(522) for endogenous proteolysis and residues Lys(524) and Phe(525) for the trypsin-generated peptide. This 37-kDa polypeptide is 1.9 times more active than the 98-kDa protein when assayed with the synthetic trisaccharide, alpha-D-Glc1,2alpha-D-Glc1,3alpha-D-Glc-O(CH2)(8)COOCH(3), and is not glycosylated. Identification of this relatively small fragment with catalytic activity will allow mechanistic studies to focus on this critical region and raises interesting questions about the relationship between the catalytic region and the remaining polypeptide.
    Preview · Article · Jan 2006 · Glycobiology
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    • "The structural organization of glucosidase I gene shows a relationship with the functional domains of the enzyme. The first exon codes for the cytoplasmic tail and transmembrane domain of the enzyme, the second and third exons encode the amino acid residues 116 through 192 and 193 through 257, respectively, whereas the fourth exon encodes the putative catalytic domain containing both the active and the glycosylation sites (Shailubhai et al., 1991; Pukazhenthi et al., 1993; Romaniouk and Vijay, 1997). At present, no clear function can be assigned to second and third exons. "
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    ABSTRACT: Glucosidase I initiates the processing of asparagine (N-) linked glycoproteins by removing the distal alpha1,2-linked glucosyl residue of the tetradecasaccharide Glc(3)Man(9)GlcNAc(2). The gene encoding this enzyme was isolated and its structural organization and promoter activity determined. The major transcript for glucosidase I on northern blot appeared to be 3.1 kb; Southern blotting and DNA sequencing indicated the size of the gene to be 6.8 kb, comprising four exons separated by three introns. The first exon encodes the cytoplasmic tail and transmembrane domain; the fourth encodes the putative catalytic domain of the enzyme. Exon-intron junctions are flanked by consensus splice donor and acceptor sequences. Transcription initiation sites were mapped by primer extension, ribonuclease protection assay and RT-PCR analysis. Primer extension results showed multiple initiation sites at -150, -156, and -272 bp relative to the translation initiation codon ATG. Sequence analysis of 5' flanking region showed no canonical TATA box, a high GC content, Sp1 and ETF binding sites (typical of a housekeeping gene promoter). Also noteworthy, the promoter region contains several generic STAT factor binding sites, one nearly perfect, and two half GR binding elements. Other cis- acting elements recognized by transcription factors such as AP-2, NF-kappaB, estrogen receptor, and progesterone receptor (PR) were also present in the putative promoter region. To determine the promoter activity, a construct encompassing the region between -2114 to -5 bp of the putative promoter was ligated to the chloramphenicol acetyltransferase (CAT) reporter plasmid and transiently transfected into COS 7 cells. CAT assay results clearly show transcriptional activity of the promoter.
    Preview · Article · Sep 1999 · Glycobiology
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    ABSTRACT: As the enzyme that initiates the maturation phase of the oligosaccharide moiety of N-linked glycoproteins, glucosidase I controls the flux of carbohydrate during the biosynthesis of these proteins. In a previous study to elucidate the structure-function relationships, we reported the presence of a cysteine residue at or near the active site of the enzyme from the bovine mammary gland (Pukazhenthi,B.S., Muniappa,N. and Vijay,I.K., 1993, J. Biol. Chem., 268, 6445-6452). We have now extended this approach to identify the participation of an arginine and a tryptophan residue in the enzyme that may play an important role in binding the substrate. The data have been combined with the results of the previous study and the cDNA-derived sequence to propose a ERHLDLRCW motif in the active site of the enzyme in the rat mammary gland that is involved in binding the incipient glycoprotein substrate for processing.
    Preview · Article · May 1997 · Glycobiology
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