| Summary: | Saccharomyces cerevisiae processing a-glucosidase I (Cwh41p) is a type II
membrane-bound glycoprotein encoded by CWH41. This enzyme regulates the first
trimming step in the A-glycosylation pathway and may play an important role in glycoprotein
biosynthesis and quality control in the endoplasmic reticulum (ER). Despite its importance,
there is a limited understanding of the structure, functional residues, and mechanism of aglucosidase
I. Therefore this thesis was focused on establishing a robust purification method
for the soluble form of yeast a-glucosidase I, identifying the catalytic domain, and
determining structural or functional amino acid residues.
The soluble form of a-glucosidase I was purified to 95% homogeneity using a
combination of ammonium sulfate precipitation, anion-exchange, lectin affinity, and sizeexclusion
chromatographies. The molecular mass of soluble a-glucosidase I was 98 kDa by
SDS-PAGE. The purification method was improved by cultivation of transformed yeast in a
fermenter and using a deoxynojirimycin (DNM)-based column. This method reproducibly
yielded 40 pg of pure enzyme per gram of wet biomass with no detectable contamination by
other aryl a- and P-glucosidases. Cleavage between Ala24 and Thr25 of the transmembrane
domain of Cwh41p released the soluble activity and this fragment was shown to be
glycosylated.
A luminal 37 kDa non-glycosylated polypeptide was isolated as the smallest active
fragment from endogenous and trypsin hydrolysis of the soluble a-glucosidase I, using
DNM-based resins. The hydrolysis sites were determined to be between Arg521 and Thr522
for endogenous proteolysis and Lys524 and Phe525 for the trypsin hydrolysis. This 37 kDa
polypeptide was 1.9 times more active than the 98 kDa protein when assayed with the
synthetic trisaccharide.
Site-specific chemical modification of the soluble a-glucosidase I from yeast using
diethylpyrocarbonate, tetranitromethane and 3-(3-(dimethylamino)propyl)carbodiimide
revealed that histidine, tyrosine and carboxylic acid residues are involved in a-glucosidase I
activity, as these residues could be protected from modification using the competitive
inhibitor DNM. DNM could not prevent inactivation of enzyme treated with N -
bromosuccinimide used to modify tryptophan residues.
Functional expression of truncated forms of Cwh41p was also investigated. Only
Cwhtlp (E35- F833) was expressed as a catalytically active soluble fragment. Cwhtlp was
isolated as a 94 kDa non-glycosylated polypeptide with a specific activity (3600 U/mg of
protein) comparable to the soluble a-glucosidase I (3000 U/mg of protein). These results
suggest that the Ml-128 region, containing the predicted N-terminal cytosolic segment and
transmembrane domain, of Cwh41p likely carries an ER-targeting signal sequence and is not
important for protein folding.
Alignment of orthologs indicated that six highly conserved carboxylic acid residues
resided within the putative catalytic region of yeast a-glucosidase I. Substitution with A la
for E580 and D584 of Cwhtlp (E613 and D617 of Cwh41p), that are situated at the
corresponding proposed binding motif of the mammalian enzyme, resulted in undetectable aglucosidase
I activity. Furthermore, mutants were expressed at considerably lower
concentrations than Cwhtlp. These findings suggest that conserved E613 and D617 may
play an important functional or structural role in enzyme activity.
In conclusion, results of this thesis indicated that the soluble a-glucosidase I is a
proteolytic product of Cwh41p and can be functionally expressed without undergoing Nglycosylation.
Moreover, the catalytically active fragment (F525-F833) can be isolated from
the soluble a-glucosidase I but it can not be expressed alone suggesting an integral structural
function for the non-catalytic region (E53-F525). Also, chemical modification results
suggest that the potential binding residues are more conserved between yeast and plant,
rather than yeast and mammalian a-glucosidase I. Finally, the conserved E613 and D617
may play an important role in yeast a-glucosidase I activity. === Land and Food Systems, Faculty of === Graduate
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