Q 1993 by The American Society for Biochemistry and Molecular Biology. Inc.
Vol. 268, No. 9, Issue of March 25, pp. 64454452,1993
Printed in U.S.A.
Role of Sulfhydryl Groups in the Function of Glucosidase I from
(Received for publication, September 10, 1992)
Budhan S. Pukazhenthi, Nagaraja Muniappa, and Inder
From the Department of Animal Sciences and the Center for Agricultural Biotechnology, University of Maryhnd,
College Park, Maryhnd 20742
Glucosidase I initiates the processing of asparagine-
linked glycoproteins by excising the distal al,a-linked
glucosyl residue from the GlcSManeGlcNAcz oligosac-
charide, soon after its en bloc transfer from the lipid-
linked donor to the nascent polypeptide. 1-Deoxynoji-
rimycin, an analog of D-glucose,
inhibitor of the enzyme. Sulfhydryl-seeking reagents
also strongly inhibit the enzyme, implying the
ment of an -SH group in its activity. To test this hy-
pothesis, glucosidase I was purified from the rat
mary gland and its active site was loaded with 1-
deoxynojirimycin, to protect such a group($,
-SH groups on the remaining surface of the enzyme
were blocked with N-ethylmaleimide orpara-chlorom-
ercuriphenylsulfonic acid. Deoxynojirimycin was re-
moved by dialysis to expose the
group(+ This group(s) w a s then tagged with 3-(N-
maleimidopropiony1)biocytin (MPB) and detected
1a61-streptavidin on Western blots. A series of experi-
ments is presented to show that indeed a critical -SH
group(s) is located within the catalytic site of the en-
zyme. Additionally, the enzyme also possesses one or
more sulfhydryls and disulfide bonds in
structure. The experimental approach outlined here
should apply to identify reactive sulfhydryl groups in
other catalytically active proteins.
is a potent competitive
active site -SH
The biosynthesis of asparagine-linked glycoproteins begins
with the assembly of the tetradecasaccharide-lipid, Glcal +
2Glcal + 3Glcal 3 3Manal + 2Manal + 2Manal 3 3
(Manal 3 2Manal3 6[Manal+ 2Manal+ 31Manal +
6)Man/3l+ 4GlcNAcBl+ 4GlcNAc-P-P-dolichol in the en-
doplasmic reticulum. After an en bloc, cotranslational transfer
to the acceptor polypeptide on the lumenal face of the ER,‘
the oligosaccharide is extensively modified as the newly syn-
thesized glycoprotein traverses the secretory pathway en route
to its final destination in an intracellular compartment or
export out of the cell (1,2).
* This work was supported by National Institutes of Health Grant
DK 19682 and Project ANI-92-10 from the Maryland Institute of
Agriculture and Natural Resources. This paper is Scientific Article
A6374, Contribution 8558 of the Maryland Agricultural Experiment
Station. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
$To whom correspondence should be addressed. Tel.: 301-405-
1407; Fax: 301-314-9921.
The abbreviations used are: ER, endoplasmic reticulum; PAGE,
polyacrylamide gel electrophoresis; DNM, 1-deoxynojirimycin; Dl”,
dithiothreitol; NEM, N-ethylmaleimide; MPB, 3-(N-maleimidopro-
piony1)biocytin; pCMPSA, para-chloromercuriphenylsulfonic acid;
An ensemble of enzymes is involved in the post-translation
modifications of the N-linked oligosaccharide on the glyco-
protein. The processing reactions begin with the action of
glucosidase I that clips the distal a1,Z-linked glucose from the
oligosaccharide, even while the glycoprotein is still bound to
the polysome (3-12). Next, glucosidase I1 in the ER excises
the two inner a1,3-linked glucosyl residues (13-20). The glu-
cosy1 moieties may also be removed by the action of a Golgi-
localized endomannosidase in an alternate, bypass pathway
in which GlcsMan, Glc2Man, and GlclMan have been shown
to be released from the a1,3 arm of the branched precursor
(21-25). Othe;. glycosidases, resident in the ER, and different
subcompartments of the Golgi that participate in the process-
ing of glycoproteins include an ER a-mannosidase (26-29), a
Man9-mannosidase (30, 31), Golgi mannosidases I (32-33)
and I1 (34, 35), and the al,3/al,6-specific mannosidase (36).
An entire spectrum of glycosyltransferases in the Golgi and
trans-Golgi network (37, 38) whose expression can be tissue-
and development-specific (39) are responsible for the incor-
poration of antennary and bisecting sugars viz., N-acetylglu-
cosamine, galactose, fucose and sialic acid, that is character-
istic of complex and hybrid glycoproteins.
The Golgi-localized glycosyltransferases have been exten-
sively investigated, and many of these have been purified and
characterized from different tissues; cDNAs have been iso-
lated encoding some of these enzymes (39-51); also the orga-
nization and the regulation of the genes of some of the
enzymes has been reported (52-59). In contrast, much less is
known about the processing glycosidases. Among these, the
ER a-mannosidase in rat liver (34, 35) and yeast (29, 60),
microsomal al,2-mannosidase (32,33), and the Golgi a-man-
nosidase I1 (61,62) have been studied in some detail.
Several laboratories have focused on the characterization
of glucosidase I and 11. Glucosidase I1 has been purified and
characterized in pig kidney (13), rat liver (15), bovine mam-
mary gland (171, mung bean seedlings, and soybean cells (20).
Immunoelectron microscopic procedures localized the enzyme
in the ER and autophagosomes (14). The purification and
characterization of glucosidase I has been reported from yeast
(4,6), liver (5,8, lo), mammary gland (9, 12), and mung bean
seedlings (7). While the yeast enzyme purified as a 95-kDa
polypeptide, the enzyme from other animal tissues appears to
be a tetramer of 320-350 kDa with a subunit molecular mass
of 85 kDa. The rat mammary enzyme was shown to be
modulated as a function of gland ontogeny; hormonal regu-
lation of the enzyme activity in explant cultures of the gland
was in good agreement with the glycosylation of a-lactalbu-
min, an asparagine-linked glycoprotein, and a characteristic
marker for the lactating tissue (11). More recently, we showed
that the polypeptide of glucosidase I consists of two contig-
uous domains: a membrane-bound domain that anchors the
protein to the endoplasmic reticulum and a hydrophilic, lu-
in Glucosidase I
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