Attachment of the saccharide concatenation to proteins is one of the most important posttranslational alterations of eukaryotic proteins, particularly secreted and membrane proteins, which lead to the formation of a host of protein-bound oligosaccharides with diverse biological maps. There are five distinguishable types of sugar-peptide bonds, i.e. N- and O-glycosylated, P-mannosylated, phosphoglycation, and glypiation (Spiro, 2002).
The N-glycosidic bond between the glycan concatenation and the Asn residue in the consensus motive (Asn-X-Ser/Thr) of a protein is most prevalent among all the types of sugar-amino acid/peptide bonds (Spiro, 1973). There are more than 16 enzymes involved in assorted steps of glycosylation that occur in the endoplasmic reticulum (ER), Golgi setup, cytosol, and nucleus.
One such enzyme is processing alpha glucosidase-I (glu-I), an ER inner membrane-bound protein. α-Glucosidase I commences pairing of N-glycans by splitting the α-(1,2)-linked glucose unit from the freshly assembled Glc3Man9GlcNAc2 after en axis transportation to the nascent sequence (Asn-X-Thr/Ser) of proteins (Grinna & Robbins, 1980).
It has become apparent that the alterations that take place in the ER reflect a spectrum of maps related to glycoprotein folding, quality control, screening, degradation, and secretion. In the ER, the Glu-I along with alpha-glucosidase-II plays a major role in the protein quality control system.
Hence, any damage in this pathway leads to drastic effects on the function of the secreted glycoprotein as well as the physiology of the organism. The deficiency of glu-I in humans leads to CDG-IIb [CDG – congenital disorders of glycosylation] (De Praeter et al., 2000). Despite its critical role in the synthesis of glycoproteins, many aspects of the structure and catalytic mechanism of glu-I are still unknown.
In Saccharomyces cerevisiae, CWH41 cistron encodes glucosidase-I
Baker’s yeast, Saccharomyces cerevisiae, is a commonly used eukaryotic model organism that only possesses two types of protein glycosylation: N-glycosylation of asparagine residues and O-mannosylation of threonine and serine residues (Lehle et al. 2006). These two types of glycosylation are highly conserved from yeast to humans. Additionally, inherited disorders of glycosylation are associated with these two types of glycosylation. Hence, in the current study, baker’s yeast will be used as the model organism to analyze the catalytic mechanism and structure-function relationship of Glu-I.
In S. cerevisiae, Glu-I is encoded by the CWH41 gene (Romero et al. 1997). It catalyzes the removal of distal α-1,2 glucose residues from the Glc3Man9GlcNAc2 (G3M9GNA2) chain co-translationally attached to the Asn residue in the consensus motif Asn-X-Ser/Thr of a nascent protein (Kornfeld and Kornfeld 1985). It belongs to the glycosyl hydrolase family 63. This enzyme was first purified by Bause et al. (1986) and reported to be a 95 kDa glycoprotein with an optimal pH of 6.8. Glu-I, along with glucosidase-II…
Based on previous studies by Bause et al. (1986), it is apparent that Glu-I is a 94 kDa protein that is bound to the inner membrane of the ER. The omission of the N-terminal amino acid residues (24 residues) resulted in soluble activity (Faridmoayer). The proteolytic cleavage of Glu-I resulted in various truncated forms, among which the smallest 37 kDa fragment showed the highest catalytic activity, 1.9 times higher than that of the whole protein.
There is limited information available regarding the amino acid residues that play a role in contact action and substrate binding of Glu-I. In previous studies, site-directed mutagenesis of Glu-I was carried out (Faridmoayer and Scaman 2007). When E613 and D617 residues were replaced by alanine, Glu-I completely lost its function. This loss of function was either due to the loss of active conformation or the loss of catalytic residues.
Processing α-glucosidase I or mannosyl-oligosaccharide glucosidase (EC 188.8.131.52) is a type-II membrane-bound glycoprotein with 833 amino acids. The omission of the transmembrane region (1-24 amino acids at the N-terminus) resulted in a soluble, glycosylated, functional Glu-I, whereas the omission of the N-terminal 34 amino acids leads to the nonglycosylated catalytically active protein. This truncated catalytically active 94 kDa fragment of Glu-I may be expressed in any strong prokaryotic expression systems, like pET in E. coli, due to the absence of glycosylation, to facilitate easy purification.
In general, the hydrolysis by glycosidases occurs via two major mechanisms, giving rise to either an overall keeping or an inversion of the constellation (Koshland, 1953). The Glu-I from S. cerevisiae was found to be an inverting glycosidase (Palcic). Understanding the mechanism of Glu-I can assist in the development of mechanism-based drugs. The hydrolysis of the glycosidic bond by glycosidases takes place through acid-base contact action, which requires two amino acids where one serves as a proton donor and the other as a nucleophile (ref).
Based on old findings on Glu-I (Faridmoayer and Scaman, 2007), we hypothesize that any two of the conserved carboxylic residues (D601, E613, D617, D602, D670, and E804) play an important function in either contact action or substrate binding since these residues lie in the catalytic part. However, homology studies revealed that the carboxylic residues E613 and E804 are the possible catalytic residues.
Based on current understanding, trypsin hydrolysis of Glu-I at positions Lys524 and Phe525 releases a 37 kDa fragment which is 1.9 times more active than the whole enzyme. The recombinant expression of the 37kDa fragment as an individual protein in S. cerevisiae did not lead to a catalytically active protein. This may be due to the need for the non-catalytic N-terminal domain for the active conformation/folding. Based on these studies on Glu-I, we can speculate that the N-terminal domain of Glu-I has two important functions, i.e., aid in getting an active conformation/folding and regulatory function.
We hypothesize that the N-terminal fragment, due to its regulatory function, decreases the activity by strictly specific binding to its natural substrate. The 37 kDa truncated form, which is not regulated by the N-terminal domain, may show relaxed specificity for sugars (further supporting information needed. YGJK may be an example for this, but homology). We also hypothesize that the N-terminal domain of Glu-I shows lectin-like activity, which facilitates specific binding to glycan chains.
The main obstruction in studying the structure and catalytic mechanism of Glu-I is the quantity of the active protein. Purification of ER membrane-bound Glu-I is boring and also yields are very low (ref). In order to obtain large quantities of Glu-I for further characterization studies, the overexpression of Glu-I will be carried out in E. coli since N-glycosylation is not important for the function of Glu-I.
Generally, the mechanism of action of glycosyl hydrolases involves the engagement of two carboxylic residues. Based on old findings (Faridmoayer and Scaman, 2007), we hypothesize that any two of the conserved carboxylic residues (D601, E613, D617, D602, D670, and E804) play an important function in either contact action or substrate binding since these residues lie in the catalytic part.
However, homology studies revealed that the carboxylic residues E613 and E804 are the possible catalytic residues. Using site-directed mutagenesis, amino acids at these positions will be replaced by their alkyl forms. Further mutagenesis studies will also be carried out to understand the catalytic mechanism.
The pH and temperature optima and the kinetic parameters (substrate specificity, Km, Vmax, and Ki against inhibitors like deoxynojirimycin) of the catalytically active variants will also be investigated. Functional expression of truncated forms of CWH41 will be conducted to gain further understanding of the roles of various domains.
Three-dimensional structures of eukaryotic glu-I have not yet been reported, although they have been extensively studied. The studies to determine the three-dimensional structure of glu-I are being conducted at a collaborator’s research lab. However, preparations will be made for crystallographic studies of the C-terminal 37kDa fragment and mutant forms of glu-I.
E. coli expresses a protein called YgjK which is homologous to glu-I (Kurakata et al., 2008). Due to its more relaxed substrate specificity, mutagenesis studies will also be carried out on YgjK to understand the important residues involved in substrate binding and catalytic action.
His pull-down to determine interaction between N- and C-terminal domains.
Co-immunoprecipitation is typically used to test whether two proteins interact with each other. The N- and C-terminal domains will be expressed as individual proteins. The purified His-tagged N-terminal domain will be treated with HRV3C peptidase and purified using IMAC affinity chromatography to remove its 6xHis tag. The N-terminal domain (without its His tag) thus obtained will be incubated with His-tagged C-terminal domain which is immobilized onto a Ni2+ affinity column. The bound protein will be eluted and eluted fractions will be analyzed on SDS-PAGE.
If any interaction is observed between the C-terminal and N-terminal domains, further experiments will be conducted to determine the exact location or region that is responsible for the interaction.
Determination of lectin activity of glu-I
The N-terminal part may act as a sugar-binding domain (lectin-like) and regulate the activity of glu-I. The lectin-like activity of glu-I will be determined by using microtiter plates that are impregnated with various glycan chains.
Structural characterization of discrepancies of glu-I
The structure of glu-I will be determined using crystallography and X-ray diffraction methods, which will be carried out at the affiliated lab. The crystals will be grown using the hanging drop vapor diffusion method. The crystals thus formed will be used for diffraction and subsequent analysis for the determination of the 3D structure.
The biochemical activity of glu-I is critical for the normal development and function of many organisms, including humans (De Praeter et al. 2000). Understanding various aspects of this enzyme will help in understanding numerous disease states that are directly or indirectly associated with its function.
Inhibitors of the N-linked glycosylation pathway have been extensively tested for antiviral activity since viral membrane proteins are synthesized and modified post-translationally by host machinery. The inhibitors of glu-I reportedly inhibited the reproduction of many viruses, which require heavily glycosylated proteins (that strongly depend on calnexin/calreticulin lectin chaperone system) for their assembly and function, including human immunodeficiency virus (Dwek et al. 2002).
Understanding the structure and catalytic mechanism of glu-I will significantly help in developing mechanism-based drugs that are useful in the treatment of various disease conditions. Currently, many glu-I inhibitors, such as [drug name], are used as drugs to treat viral infections. Mechanism-based inactivators/drugs provide the same potential clinical advantages as previously observed with inhibitor-based drugs.
The dissociation rate for mechanism-based drugs, which covalently modify their target enzyme, is very low or equals zero, whereas the interaction of inhibitors with their enzyme counterparts is reversible. Mechanism-based drugs rely on the chemistry of the enzyme active site and are highly selective to target enzyme, which provides ultimate specificity for use as drugs.
Understanding of glu-I will also enhance the knowledge of N-glycosylation, which is necessary for the use of glycan chains or glycoengineering for heterologous production of various medically or commercially important proteins.
The processing α-glucosidase-I plays a significant role in the quality control of protein N-glycosylation. The current study will enhance the understanding of glu-I catalytic mechanism and structural insights. The site-directed mutagenesis of glu-I will provide more details about the amino acid residues that play a role in contact action.