Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II

Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purified brush border preparations. Kidney glucosidase II contained species carrying endo H-sensitive, high mannose as well as endo H-resistant oligosaccharide chains. Some species of glucosidase II contained sialic acid. The sialylated species were enzymatically active. This study demonstrates than an enzyme presumed to be a resident of the endoplasmic reticulum may show alternative localizations in some cell types.     

Yeast GTB1 encodes a subunit of glucosidase II required for glycoprotein processing in the endoplasmic reticulum

Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose alpha-subunit contains the glycosyl hydrolase active site. The function of the beta-subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the beta-subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96-102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast alpha-subunit homologue Gls2p. Surprisingly, we found that Deltagtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.     

Promiscuous activity of ER glucosidase II discovered through donor specificity analysis of UGGT

In glycoprotein quality control system in the endoplasmic reticulum (ER), UGGT (UDP-glucose:glycoprotein glucosyltransferase) and glucosidase II (G-II) play key roles. UGGT serves as a glycoprotein folding sensor by virtue of its unique specificity to glucosylate glycoproteins at incompletely folded stage. By using various UDP-Glc analogues, we first analyzed donor specificity of UGGT, which was proven to be rather narrow. However, marginal activity was observed with UDP-galactose and UDP-glucuronic acid as well as with 3-, 4- and 6-deoxy glucose analogues to give corresponding transfer products. Intriguingly, G-II smoothly converted all of them back to Man(9)GlcNAc(2), providing an indication that G-II has a promiscuous activity as a broad specificity hexosidase.     

Isolation and functional characterization of Sporothrix schenckii ROT2, the encoding gene for the endoplasmic reticulum glucosidase II

The N-linked glycosylation is a ubiquitous protein modification in eukaryotic cells. During the N-linked glycan synthesis, the precursor Glc(3)Man(9)GlcNAc(2) is processed by endoplasmic reticulum (ER) glucosidases I, II and α1,2-mannosidase, before transporting to the Golgi complex for further structure modifications. In fungi of medical relevance, as Candida albicans and Aspergillus, it is well known that ER glycosidases are important for cell fitness, cell wall organization, virulence, and interaction with the immune system. Despite this, little is known about these enzymes in Sporothrix schenckii, the causative agent of human sporotrichosis. This limited knowledge is due in part to the lack of a genome sequence of this organism. In this work we used degenerate primers and inverse PCR approaches to isolate the open reading frame of S. schenckii ROT2, the encoding gene for α subunit of ER glucosidase II. This S. schenckii gene complemented a Saccharomyces cerevisiae rot2Δ mutant; however, when expressed in a C. albicans rot2Δ mutant, S. schenckii Rot2 partially increased the levels of α-glucosidase activity, but failed to restore the N-linked glycosylation defect associated to the mutation. To our knowledge, this is the first report where a gene involved in protein N-linked glycosylation is isolated from S. schenckii.     

Substrate specificity of cucumisin on synthetic peptides

The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leu(m)-Pro-Glu-Ala-Leu(n) (m = 0-4, n = 0-3). Neither Pro-Glu-Ala-Leu (m = 0) nor Leu-Pro-Glu-Ala (n = 0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leu(m)-Pro-Glu-Ala-Leu increased with the increase of m = 1 to 2 and 3, but was however, essentially same with the increase of m = 3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leu(n) increased with the increase of n = 0 to 1 and 2, but was essentially same with the increase of n = 2 to 3. Then, it was concluded that cucumisin has a S5-S3' subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu, Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser, Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1' position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1' positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.     

Endoplasmic reticulum glucosidase II is inhibited by its end products

The calnexin/calreticulin cycle is a quality control system responsible for promoting the folding of newly synthesized glycoproteins entering the endoplasmic reticulum (ER). The association of calnexin and calreticulin with the glycoproteins is regulated by ER glucosidase II, which hydrolyzes Glc 2Man X GlcNAc 2 glycans to Glc 1Man X GlcNAc 2 and further to Glc 0Man X GlcNAc 2 ( X represents any number between 5 and 9). To gain new insights into the reaction mechanism of glucosidase II, we developed a kinetic model that describes the interactions between glucosidase II, calnexin/calreticulin, and the glycans. Our model accurately reconstructed the hydrolysis of glycans with nine mannose residues and glycans with seven mannose residues, as measured by Totani et al. [Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) J. Biol. Chem. 281, 31502-31508]. Intriguingly, our model predicted that glucosidase II was inhibited by its nonglucosylated end products, where the inhibitory effect of Glc 0Man 7GlcNAc 2 was much stronger than that of Glc 0Man 9GlcNAc 2. These predictions were confirmed experimentally. Moreover, our model suggested that glycans with a different number of mannose residues can be equivalent substrates of glucosidase II, in contrast to what had been previously thought. We discuss the possibility that nonglucosylated glycans, existing in the ER, might regulate the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle. Our model also shows that glucosidase II does not interact with monoglucosylated glycans while they are bound to calnexin or calreticulin.     

Endoplasmic reticulum glucosidase II is required for pathogenicity of Ustilago maydis

We identified a nonpathogenic strain of Ustilago maydis by tagging mutagenesis. The affected gene, glucosidase1 (gas1), displays similarity to catalytic alpha-subunits of endoplasmic reticulum (ER) glucosidase II. We have shown that Gas1 localizes to the ER and complements the temperature-sensitive phenotype of a Saccharomyces cerevisiae mutant lacking ER glucosidase II. gas1 deletion mutants were normal in growth and mating but were more sensitive to calcofluor and tunicamycin. Mutant infection hyphae displayed significant alterations in the distribution of cell wall material and were able to form appressoria and penetrate the plant surface but arrested growth in the epidermal cell layer. Electron microscopy analysis revealed that the plant-fungal interface between mutant hyphae and the plant plasma membrane was altered compared with the interface of penetrating wild-type hyphae. This may indicate that gas1 mutants provoke a plant response.     

Specific isoforms of the resident endoplasmic reticulum protein glucosidase II associate with the CD45 protein-tyrosine phosphatase via a lectin-like interaction

We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic alpha-chain and an associated beta-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the alpha-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GIIalpha is capable of associating with CD45 in a competitive situation where multiple GIIalpha isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GIIalpha acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation.     

Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides

The specificity of casein kinase II has been further defined by analyzing the kinetics of phosphorylation reactions using a number of different synthetic peptides as substrates. The best peptide substrates are those in which multiple acidic amino acids are present on both sides of the phosphorylatable serine or threonine. Acidic residues on the NH2-terminal side of the serine (threonine) greatly enhance the kinetic constants but are not absolutely required. Acidic residues on the COOH-terminal side of the serine (threonine) are absolutely required. One position for which the occupation of an acidic residue is especially critical is the position located 3 residues to the COOH terminus of the phosphate acceptor site, although the presence of an acidic amino acid in the positions that are 4 or 5 residues removed may also provide an appropriate structure that will serve as a substrate for the kinase. Aspartate serves as a better amino acid determinant than glutamate. A relatively short sequence of amino acids surrounding the phosphate acceptor site appears to serve as the basis for the specificity of casein kinase II. The peptides in this study were also assayed with casein kinase I and the casein kinase from the mammary gland so that the specificities of these kinases could be compared to that of casein kinase II.     

Substrate specificity and diastereoselectivity of strictosidine glucosidase, a key enzyme in monoterpene indole alkaloid biosynthesis

Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady-state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted.     

Protein-mediated phospholipid translocation in the endoplasmic reticulum with a low lipid specificity

The outside-inside translocation rate of various amphiphilic spin-labeled phospholipids has been measured in rat liver endoplasmic reticulum vesicles. The eight spin-labels tested experienced a fast flip-flop rate with the same half-time of approximately 20 min at 37 degrees C. The stationary distribution of these phospholipid analogues was ca. 45% on the inner vesicular leaflet and 55% on the external one, showing that there is no net enrichment of some lipid in one layer under the experimental conditions used. The initial rate of translocation was reduced 4-fold if membranes were preincubated with N-ethylmaleimide (2 mM) and was about an order of magnitude lower in liposomes made from the extracted lipids. An apparent saturability of the transbilayer diffusion can be deduced from the variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated in the membrane. Moreover, translocation rates of two different spin-labeled phospholipids introduced simultaneously in the membrane were almost equally reduced by the presence of the other lipid. On the other hand, no competition between the water-soluble dibutyroylphosphatidylcholine and the amphiphilic spin-labeled phospholipids could be detected. Overall, these results suggest that phospholipid translocation in the endoplasmic reticulum is a protein-mediated process with a low specificity, which tends, in the absence of any other metabolic event, to equilibrate the phospholipid composition of the two membrane halves.     

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.     

Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.     

Changing the specificity of the sorting receptor for luminal endoplasmic reticulum proteins.

Luminal proteins of the endoplasmic reticulum (ER) share a common carboxy-terminal tetrapeptide which is necessary and sufficient for their retention in the ER. In animal cells this retention signal is usually KDEL, whereas the yeast Kluyveromyces lactis uses the closely related sequences HDEL and DDEL. The yeast ERD2 gene has been shown to determine the capacity and specificity of the retention system, implying that it encodes a sorting receptor. This receptor is thought to retrieve escaped ER proteins from the Golgi, where a human homologue of this protein has been located. This dual function of binding and retrieval requires a receptor with highly specific binding at a specific location in the cell (Golgi but not ER). Here, a region of the ERD2 protein responsible for the specificity of ligand recognition has been identified using three independent approaches. A single amino acid residue is shown to selectively affect HDEL retention: substitution of residue 51 of the K. lactis receptor is sufficient to abolish recognition of HDEL but not DDEL, generating a novel retention phenotype.     

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.     

Structural elements in dextran glucosidase responsible for high specificity to long chain substrate

Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta-->alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar kcat/Km value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta-->alpha loop 4 of B. subtilis oligo-1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta-->alpha loop 4 were important for high activity to long-chain substrates.     

Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine-linked oligosaccharides

Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific alpha-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 microM, 20 microM, 12 microM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 1-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was approximately equal to 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 degrees C in the presence of detergent (t 1/2 approximately equal to 20 days) and showed high specificity for the hydrolysis of the terminal (alpha 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.