Glycobiology Advance Access originally published online on June 9, 2004
Glycobiology 2004 14(10):909-921; doi:10.1093/glycob/cwh110
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Glycobiology vol. 14 no. 10 © Oxford University Press 2004; all rights reserved.
Processing enzyme glucosidase II: proposed catalytic residues and developmental regulation during the ontogeny of the mouse mammary gland
2 Department of Animal and Avian Sciences, University of Maryland, College Park MD 20742; 3 Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park MD 20742
Received on April 2, 2004; revised on June 3, 2004; accepted on June 4, 2004
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Following the action of glucosidase I to clip the terminal
1,2-linked glucose, glucosidase II sequentially cleaves the two inner 1,3-linked glucose residues from the Glc1,2Glc1,3Glc1,3Man9GlcNAc2 oligosaccharide of the incipient glycoprotein as it undergoes folding and maturation. Glucosidase II belongs to family 31 glycosidases. These enzymes act by the acid-base catalytic mechanism. The cDNA of the wild-type and several mutant forms of the fusion protein of the enzyme in which mutations were introduced in the conserved motif D564MNE567 were expressed in Sf9 cells, and the proteins were purified on Ni-NTA matrix. The catalytic activity of the purified proteins was determined with radioactive Glc2Man9GlcNAc2 substrate. The results show that the aspartate and glutamate within the D564MNE567 motif can serve for catalysis, most likely as the acid-base pair within the active site of the enzyme. The developmental regulation of glucosidase II was studied during the ontogeny of the mouse mammary gland for its growth and differentiation. The mRNA of both and ß subunits of the enzyme, immunoreactive and ß subunits, and enzyme activity were measured over the complete developmental cycle. The changes in all the parameters were consistent with similar fluctuations with several other enzymes of the N-glycosylation machinery reported earlier, reaching a three- to fourfold increase over the basal level in the virgin gland at the peak of lactation. Altogether it appears that there is a coordinated regulation of the enzymes involved in protein N-glycosylation during the development of the mouse mammary gland. Key words: active site / glucosidase II / mammary gland
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Protein N-glycosylation in eukaryotic cells is initiated by the synthesis of a lipid-linked carbohydrate precursor, Glc3Man9GlcNAc2-P-P-Dol, in the dolichol cycle. After an en bloc transfer of the oligosaccharide to the nascent polypeptide in rough endoplasmic reticulum (RER) (Kornfeld and Kornfeld, 1985
; Moremen et al., 1994), the carbohydrate moiety undergoes extensive remodeling that begins with the removal of distal 1,2-linked glucose, catalyzed by glucosidase I (Glu). Glucosidese II (GluII) then removes the middle 1,3-linked glucose on the incipient glycoprotein. The resulting monoglucosylated glycan is recognized by the lectin chaperones calnexin and calreticulin, which together with ERp57 facilitate the folding and maturation of the glycoproteins in the RER. Eventually, glucosidase II removes the innermost glucose residue from the oligosaccharide in preparation for egress of the glycoprotein from the RER (Ellgaard and Helenius, 2001). Incompletely folded glycoprotein is recognized by the ER-localized UDP-Glc:glycoprotein glucosyltransferase, reglucosylated and returned for folding on rebinding by calnexin or calreticulin and additional action of Glu II (Helenius and Aebi, 2001).
Analysis of highly purified preparations of glucosidase II has shown that the enzyme is a heterodimeric protein containing a catalytic glycopeptide of 107–116 kDa ( subunit) and a smaller subunit of 58–80 kDa (ß subunit). The subunit is a soluble protein with one glycosylation site carrying a high-mannose oligosaccharide. The ß subunit has the ER retention signal (HDEL) at its C-terminus (Arendt and Ostergaard, 1997; Flura et al., 1997; Trombetta et al., 1996; Trombetta et al., 2001). It appears that the ß subunit provides the signal to retain the enzyme in the ER (Lucocq et al., 1986).
The primary amino acid sequence of the subunit of the enzyme, especially the region in its middle and C-terminal part, bears significant homology to the sequences of lysosomal -glucosidase, sucrose-isomaltase, and enzymes in family 31 glycosidases (Henrissat and Davis, 1997; Henrissat and Romeu, 1995). The amino acid sequence of the subunit, deduced from its cDNA contains the well-conserved motif (G/F)(L/I/V/M)WXDMNE, present in several of the enzymes. Notably, the motif has aspartate (D) and glutamate (E) as potential residues for acid-base–mediated catalysis by these enzymes. The present investigation was undertaken to test the hypothesis that the acid-base pair required for the catalytic action of the enzyme may be contained within the DMNE portion of the motif. The cDNAs of the wild-type mouse enzyme and site-specific mutants within the motif were expressed in insect Sf9 cells and analyzed for catalytic activity. The results support the view that aspartate564 and glutamate567 can serve to hydrolyze the substrate and as the acid-base pair for the activity of the enzyme.
The mammary gland offers an excellent model for studying the biosynthesis and developmental regulation of N-linked glycoproteins. Mammogenic and lactogenic hormones intensively modulate the development and differentiation of the gland as it repeatedly cycles between dormancy, growth, and differentiation on pregnancy, lactation, and postlactational regression. During lactation, the mammary gland synthesizes significant levels of N-linked glycoproteins, such as -lactalbumin, transferrin, and a number of proteins in the milk fat globule membrane (Keenan, 2001; Mather and Keenan, 1998).
Earlier reports from our laboratory have shown that Glu I in rat (Shailubhai et al., 1990) and three key glycosyltransferases (UDP-GlcNAc:Dol-P GlcNAc-1-P transferase, GDP-Man:Dol-P mannosyltransferase, and UDP-Glc:Dol-P glucosyltransferase) in mouse mammary glands (Vijay and Oka, 1986) are regulated parallely during gland ontogeny.
The second part of this study was focused on the developmental regulation of Glu II in the mouse mammary gland. It was considered that the developmental study of the enzyme should provide additional information on the overall regulation of the N-glycosylation machinery during the growth and differentiation of the gland. For this, the mRNA level, enzyme activity, and immunoreactive protein level of the enzyme were examined in the mouse gland at different stages of development. The results show that Glu II reaches its peak level of all of the above parameters in the mid- or late lactation stages, like the N-glycosylation enzymes reported earlier, and shows an up-regulation of 2.5–4-fold at mid- to late lactation.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Recombinant expression of catalytically active Glu II and active site analysis
Expression and affinity purification of Glu II fusion protein in Sf9 cells
The full-length cDNAs of Glu II
and ß subunits were incorporated into pBlueBacHis2 vector for expression in Sf9 cells. The construction was such that on expression, the His6 tag at the N-terminus of both subunits of Glu II would be cleaved along with the signal peptide. The subunit would be expressed as a fusion polypeptide with myc-His6 tag at its C-terminus, whereas the ß subunit with the HDEL motif at the C-terminus would be expressed without any tag.
The recombinant glucosidase II was expressed in Sf9 cells by coinfections of Glu II- and Glu II-ß recombinant viruses in a 1:1 ratio. The enzyme activity above the background level could be detected in cell lysates 24 h postinfection. It reached a maximum at 84 h and began to decline thereafter (data not shown). Coinfection of both and ß subunit was necessary for optimal activity of the recombinant Glu II. When expressed alone, the fusion polypeptide of subunit was distributed in both the membrane fraction as well as the soluble fraction and showed only 
70% activity of the heterodimeric -fusion/ß subunit recombinant protein (not shown).
To investigate the effect of mutagenesis of the potential catalytic amino acids at the active site of the recombinant enzyme, the fusion enzyme was purified using Ni-NTA agarose beads. Two bands detected in the eluted fraction (Figure 1A) represent the fusion proteins corresponding to Glu II--MycHis6 and ß subunit with molecular weight of 123 kDa and 80 kDa, respectively. Only the subunit was detected in the western blot with anti-His6 antibody (Figure 1B), because the ß subunit was designed to be expressed without the His6 tag.
|
To confirm the identities of proteins in the eluted fraction, western blots with anti-Glu II-
and anti-Glu II-ß antibodies were performed. The subunit was recognized by both anti-His6 antibody (Figure 2A) and anti-Glu II- antibody (Figure 2B). The ß subunit was recognized only by anti-Glu II-ß antibody in the eluted protein (Figure 2C). The minor bands with lower molecular weight in anti-Glu II-ß blot were assumed to be the degradation products of the ß subunit; these were also observed in previous studies (Arendt and Ostergaard, 1997; Flura et al., 1997; Trombetta et al., 1996; Pelletier et al., 2000). The results indicate that the two major protein bands in the eluted fraction are indeed the Glu II--MycHis6 fusion protein and Glu II-ß. The interaction between the fusion form of and ß subunits is specific and strong enough to sustain the purification procedure.
|
For activity assays, two controls were employed, viz. lysate from uninfected Sf9 cells and lysate from Sf9 cells that were transfected with the XylE-pAcHLT plasmid that encodes the bacterial protein XylE with His6 tag. The purification procedure removed all the endogenous enzyme activity in the supernatant. The results presented in Table I show that the control eluates from the Ni-NTA agarose beads have no Glu II activity, wheras the fusion protein presented a specific activity of 2170 ± 216 U/mg protein. The enzyme activity in the eluted fusion protein is inhibited >90% by 20 mM 1-deoxynojirimycin (DNM). Not surprisingly, the recombinant form of the enzyme has no catalytic activity toward Glc3Man9GlcNAc2, substrate for Glu I (Table I).
|
The recombinant Glu II is an N-linked glycoprotein
The sequence of glucosidase II
subunit has one potential site for N-linked glycosylation, at asparagine97. The native enzyme is an N-linked glycoprotein with a high-mannose sugar chain. To investigate whether the fusion form of the recombinant enzyme was glycosylated in the insect cells, the purified fusion enzyme was treated with endoglycosidase H (Endo H). This treatment caused a decrease in the size of subunit and a shift in the mobility on electrophoresis by 2 kDa on 7.5% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) (Figure 3A).
|
When the Endo H–treated and untreated enzyme samples were applied to Conacanavalin A (Con A)–Sepharose 4B column, the untreated enzyme was retained in the Con A–Sepharose column, with no protein detected in the flow-through sample. In contrast, the Endo H–treated enzyme ran through the column with majority of the protein recovered in the flow-through sample, although the mobility shift was not distinguishable by 10% SDS–PAGE.
The results indicate that the recombinant fusion form of the enzyme is expressed as an N-linked glycoprotein with one high-mannose oligosaccharide moiety, most likely attached to asparagine97. Apparently, the fused Myc-His6 tag at the C-terminus of the subunit does not affect N-glycosylation of the polypeptide during biosynthesis.
The substrate-binding motif in Glu II
Over 2000 glycosidases are classified into 90 families, with each family using the same catalytic mechanism, retaining or inverting, and the essential catalytic amino acid residues well conserved. Sequence analysis assigned Glu II to family 31 (Flura et al., 1997), containing the (G/F)(L/I/V/M)WXDMNE motif as highly conserved segment in all the family members (Frandsen and Svensson, 1998).
Previous studies showed that Conduritol B epoxide reacted with the conserved putative catalytic aspartic acid residue within DMNE motif in four different family 31 enzymes (Hermans et al., 1991; Iwanami et al., 1995; Kimura et al., 1997; Quaroni and Semenza, 1976). The same aspartic acid residue in the motif was also identified as the catalytic nucleophile at the active site of another family member, Aspergillus niger -glucosidase with the labeling reagent, 5-fluoro--D-glucopyranosyl fluoride (Lee et al., 2001). In addition, the mutant Asp518
Asn/Glu,Gly of human lysosomal -glucosidase (Hermans et al., 1991) and Asp470Gly of S. occidentalis glucoamylase (Kimura et al., 1997) are inactive. This invariant aspartic acid residue in the DMNE motif was suggested to be the catalytic nucleophile (Herman et al., 1991; Kimura et al., 1997; Lee et al., 2001), and the results on mutants strongly support that it is essential in catalysis.
A mining of the new information in the genomic databank, as presented in Figure 4, shows a nearly perfect homology in the region around this motif in higher eukaryote Glu II and an excellent similarity with the region in A. thaliana and S. cerevisiae. These observations strongly support the view that D564MNE567 in the sequence of the mouse enzyme may be part of the substrate-binding region within the active site cavity of the enzyme.
|
Catalytic amino acid residues in the
subunit of Glu IIA total of nine mutants of D564MNE567 motif in the
subunit of the fusion form of the enzyme were constructed. The mutants were expressed in Sf9 cells by coinfection of mutant and wild-type Glu II-ß baculoviruses. After purification, the catalytic activity was measured with the wild-type and mutant forms of the fusion enzyme (Table II). Mutations D564N, E567Q, and the scramble mutation YVWNDMNEEDYMWNVN lowered the activity of Glu II fusion protein to undetectable level of the control. Mutations D564E and E567D retained >40% and >50% of the wild-type enzyme activity, respectively. The double-switch mutation, D564EMNE567D, and mutations DM565AN566AE, D564EM565AN566AE567D, and F571A retained about 70% of the enzyme activity. These results strongly support the view that aspartate564 and glutamate567 are critical residues within the substrate-binding motif; the removal of charge on one or both aspartate564 and glutamate567 markedly lowers the enzyme activity of the expressed protein. Apparently, the active site cleft of the enzyme provides flexibility and a dynamic interaction with the substrate such that the exchange of aspartate and glutamate within DMNE does not significantly affect the activity of the enzyme. In contrast, neither the wild type nor the different mutants could catalyze the hydrolysis of terminal glucose from Glc3Man9GlcNAc2, the substrate for Glu I (data not shown). In another set of controls, 20 mM DNM added to preparations of the wild-type enzyme and different mutants inhibited the glucosidase II activity by >90%.
|
The observations of changes in enzyme catalytic activity could be considered equivocal and explained on the basis that these mutants might not have been expressed or might have been degraded as malfolded proteins after expression. Samples of the purified fusion proteins employed in the experiment shown in Table II were immunobloted with various antibodies (Figure 5). The results showed that all proteins were expressed at similar level and the mutations did not affect the interaction between
and ß subunits.
|
With the evidence that aspartate564 and glutamate567 are essential residues for Glu II activity, the kinetics and DNM inhibition experiments were focused on the mutants of these two amino acids, namely, the mutants D564N, D564E, E567Q, and E567D. The wild-type and mutant fusion proteins were incubated with substrate for various times (Figure 6). The wild-type fusion protein consumed nearly 60% of the substrate over the 2-h time period, with a nearly linear rate for the first 30 min. Mutants D564N and E567Q did not show any measurable activity during this incubation time. Mutants D564E and E567D consumed only 40% and 30% of the substrate, respectively. This agrees with the lower catalytic activity of the mutant enzyme fusion proteins compared to the wild-type fusion protein. The enzyme activity seemed to slow down gradually after incubation at 37°C and reach a plateau, possibly due to the instability of the fusion proteins. When a fresh aliquot of the wild-type enzyme was added to the reaction after 2 h of incubation, the substrate was rapidly depleted at a linear rate in the next hour (data not shown).
|
DNM is a well-known inhibitor of Glu II with an IC50 of around 5 mM. In the inhibition experiment, DNM inhibited 50% of D564E and E567D mutants' activities at 2–3 mM (Figure 7).
|
Developmental regulation of Glu II in mammary gland
mRNA level of Glu I and II measured by quantitative RT-PCR
Equal amounts of total RNA from each sample were used for the reverse transcription (RT) reaction, with oligo(dT)20 as the primer. Aliquots of the RT reaction mixture were then used as the templates for polymerase chain reaction (PCR) to amplify the specific cDNA with gene-specific primer sets (Table III) in the presence of [32P]dCTP.
|
Both Glu II-
and Glu II-ß were amplified to give a single product of 860 bp and 390 bp, respectively (Figure 8A). When ß-actin mRNA levels were determined to normalize the data, both Glu II- and Glu II-ß mRNA levels increased gradually throughout gestation and into early lactation (lactation day 7). The peak value was reached and maintained from lactation day 11 until the end of lactation (day 21). Compared to the level in the virgin animals, there was a fourfold increase in the mRNA level (Figure 8B). After weaning, the mRNA level dropped dramatically back to the base level in virgin animals within 2 days. The level dropped even lower by the 7th day after weaning. The glucosidase I mRNA levels also present a similar pattern during the tissue differentiation.
|
Enzyme activity of Glu II during different stages of mammary gland development
Microsomal fractions were prepared from different samples as described earlier (Viajy and Oka, 1986
). The enzyme was extracted from the microsomes with detergent (Triton X-100), and its activity was determined with [Glc-3H]Glc2Man9GlcNAc2. The specific activity was determined per tissue weight, total protein, and total DNA (Figure 9). The specific activity increased continuously from early gestation until midlactation. The peak value was reached around lactation day 14, with a fourfold increase compared to the background level in virgin animals. The activity dropped back to background levels soon after weaning. With the total DNA level representing the cell number, the enzyme activity was two- to threefold higher in the midlactating than in the virgin tissues. These results indicate that the significant increase in Glu II enzyme activity is not simply a result of gland growth and a rapid proliferation of the epithelial cells during gestation and lactation, but rather the enzyme activity is stimulated in the cells as the tissue undergoes differentiation.
|
Protein expression level of Glu II
The protein level of Glu II was measured by immunoblots using specific antibodies to the Glu II-
and Glu II-ß subunits. Equal amounts of total protein from each sample was subjected to 10% SDS–PAGE, followed by immunoblotting. The major bands with expected molecular weights were detected in the blots (Figure 10A). Both Glu II- and Glu II-ß protein levels present a similar pattern of changes during gland development as the enzyme activities (Figure 10B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Glu II plays a key role during the folding and maturation of the incipient N-linked glycoproteins in the RER of the secretory pathway in the eukaryotic cell. This study was undertaken with two objectives: (1) to gain an insight into catalytic residues at the active site of the enzyme, and (2) examine its regulation during the development of the mouse mammary gland as it completes the cycle from virgin
gestationlactationpostlactational involution during growth and differentiation.
Glu II is a heterodimeric protein: Its subunit possesses the catalytic activity, and the ß subunit has the HDEL signal at the carboxyl terminal, characteristic for the retention of proteins in the RER. Alignment of the amino acid sequence of the subunit of the mouse enzyme deduced from the cDNA assigned it to family 31 glycosidases (Flura et al., 1997; Frandsen and Svensson, 1998). The glucosidases in this family hydrolyze their substrates by acid-base catalysis and release -glucose with retention of configuration at the anomeric carbon. Aspartate and glutamate have been shown as the catalytic residues in these enzymes. Glycosyl-enzyme conjugates with 5-fluoro--D-glucopyranosyl fluorides have served to trap the intermediate for identifying the catalytic nucleophilic amino acid at the active site of retaining glycosidases (Lee et al., 2001). Conduritol B epoxide has also been used to label the nucleophile, although its labeling is not very specific (Kimura et al., 1997)
A number of family 31 glycosidases contain a highly conserved region, (G/F)(L/I/V/M)WXDMNE, which is also found in the subunit of Glu II. The aspartate and glutamate within the DMNE segment are believed to serve as the acid-base catalysts. The lysosomal -glucosidase and the sucrase-isomaltase have this motif at their active site. Thus the aspartate and glutamate residues in D564MNE567 of the mouse Glu II appeared to be good candidates to examine catalytic activity. X-ray crystallography of more than 20 glycosidases has revealed the distance between the two catalytic acid-base groups at the active site to be 4.8–5.5 Å in retaining and 9–10 Å in inverting enzymes (Withers and Abersold, 1995). A priori, the aspartate and glutamate in the motif might appear to be too close to each other for their catalytic role as a pair; however, precedents have been described for similar neighboring catalytic residues in other glucosidases. For example, aspartate170 and 172 and glutamate174 were implicated via mutagenesis to be the catalysts at the active site of ß-N-acetylglucosaminidase from Streptomyces plicatus (Schmidt et al., 1994). Likewise, aspartate200 and glutamate204, separated by only three residues, serve as the catalytic pair of chitinase in B. subtilis (Watanabe et al., 1993).
The motif in the subunit was chosen for site-specific mutagenesis of D564 and E567 to explore their potential role in catalysis by the enzyme. Conservative site-specific mutations of the residues to neutral asparagine and glutamine abolished the catalytic activity of the fusion protein of the enzyme. A scramble mutation that changed YVWNDMNE to EDYMWNVN within the motif also resulted in a total loss of the enzyme activity. On the other hand, an exchange of these residues with each other, singly as in D564E and E567D mutants, retained >40% and >50% of the catalytic activity, respectively. The double-switch mutation, D564EMNE567D, resulted in more than 70% retention of the catalytic activity of the fusion enzyme. These results support the view that aspartate564 and glutamate567 can serve to catalyze the hydrolytic reaction by Glu II.
The side chains of aspartate and glutamate differ in size by one -CH2- in length. Minimally, in a single switch, this will affect the overall steric configuration of the active site of the mutant enzyme to give either D564MND567- or E564MNE567-containing fusion proteins that are structurally different from the wild-type enzyme. The double switch mutation, D564EMNE567D, would also be expected to affect the steric geometry of the active site. The retention of a significant level of activity after these mutations in the motif shows that the active site of the enzyme is flexible; it engages in a dynamic interaction with the substrate to catalyze the cleavage of the distal 1,3-linked glucosyl residue on the oligosaccharyl moiety of the substrate. It is possible that the binding of the oligosaccharide may induce a conformational change at the active site such that the geometric juxtaposition of both residues remains favorable for catalysis. The mutations targeted at methionine565 and asparagine566, specifically, DM565AN566AE and D564EM565AN566AE567D, also did not affect the enzyme activity to any significant degree.
Phenylalanine571 in close proximity to the DMNE in the mouse enzyme is highly conserved across all the aligned sequences of family 31 glucosidases in GenBank (Figure 4). However, the mutation F571A had no effect on the activity of the expressed enzyme. Apparently, the active site of the enzyme shows a certain amount of flexibility at this residue as long as the acid-base pair residues in the motif are favorably disposed for catalysis. It is worth emphasizing that the evidence for the participation of D564 and E567 as the acid-base pair for catalysis by Glu II may be considered tentative considering that Km and kcat values on the expressed wild type and different mutant enzymes have not yet been determined; folding characteristics of the expressed enzyme would require a determination of these parameters.
Glycoprotein synthesis is associated with growth and differentiation processes. The expression of mRNA, immunoreactive levels of - and ß-subunits, and the catalytic activity of Glu II were examined during the development of the mouse mammary gland. The three parameters followed a similar pattern, reaching their highest levels of three- to fourfold around mid- and late lactational stages over the levels in the quiescent virgin gland. Glu I initiates posttranslational remodeling of the oligosaccharide of the incipient glycoprotein and acts at the step preceding the action of Glu II. Earlier we had shown that the activity and immunoreactive protein of this enzyme was regulated during the development of the mammary gland (Shailubhai et al., 1990). When mRNA levels of Glu I were examined in this study, they were found to closely parallel the changes in the levels of Glu II mRNA. These variations are almost identical to the pattern observed for the UDP-GlcNAc:Dol-P GlcNAc-1-P transferase, GDP-Man:Dol-P mannosyltransferase, and UDP-Glc:Dol-P glucosyltransferase reported previously (Rajput et al., 1994; Vijay and Oka, 1986). Apparently, the entire glycosylation machinery is coordinately modulated during the growth and differentiation of the mammary gland. The activation of the enzymes coincides with the time when the secretory activity of the mammary gland reaches peak levels.
The precise role of the ß subunit of Glu II other than its potential role in retaining the catalytic subunit in the ER via the HDEL signal is not clear. The results in Figure 10 show that there is a differential elevation in the ß subunit protein relative to the catalytic subunit. It is possible that ß subunit represents a C-terminal HDEL-containing polypeptide that serves to retrieve more soluble proteins in the ER than just the subunit of Glu II. The rapid rise and maintenance of ß subunit at the elevated level seem to persist throughout the peak of lactation and may be involved in interacting with as-yet-unidentified proteins synthesized by the lactating gland.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Materials
Anti-His6 antibody was obtained from Qiagen (Valencia, CA). Anti-Glu II-
antibody was raised as anti-peptide antibody by Research Genetics with a mixture of internal peptides of the subunit of Glu II, that is, D34RSNFKTCDESSFCKRQR51, D231KPEETQEKAEKDEPGAWEE250, and T830IVPRWMRVRRSSDCMKDD848 as antigen. Anti-Glu II-ß antibody was also raised by Research Genetics (Huntsville, AL) against a mixture of internal peptides, specifically D49QVNDDYCDCKDGSDEPGT67, E113NTCREKGRKEKESLQQ129, and Q264EARSKFEEVERSLKEMEE282 as antigen. Protein concentration was estimated with the BCA reagent according to the protocol provided by Pierce Chemical (Rockford, IL). Endo H was purchased from Glyko (Novato, CA). Other reagents were from commercial sources. The substrate oligosaccharides, [Glc-3H]Glc2Man9GlcNAc2 (oligosac 13) and [Glc-3H]Glc3Man9GlcNAc2 (oligosac 14), were prepared in our laboratory, as before (Saxena et al., 1987). The cDNA clones of mouse Glu II- (GenBank accession number AAC53182 [U92793]) and GluII-b (GenBank accession number AAC53183 [U92794]) (Arendt and Ostergaard, 1997) were the gifts of Dr. H. L. Ostergaard (University of Alberta, Edmonton). Mouse mammary glands at different developmental stages were obtained from Hilltop Laboratory Animals (Scottdale, PA).
Construction of expression vectors and recombinant baculoviruses containing Glu II cDNAs
The restriction sites for EcoRV at 5' end and HindIII at 3' end were generated by PCR, using primers 5' CGGATATCAAGATGGCGGCAATAGCG-3' and 5' CCAAGCTTTCTCGAAGATGAATACTCCT-3', respectively, from the cDNA clone of mouse Glu II-. The cDNA was ligated in-frame into the EcoRV/HindIII sites of pcDNA3. 1B(-) MycHis (Invitrogen, Carlsbad, CA), to obtain the vector Glu II--pcDNA. It contained full-length mouse Glu II- cDNA with Myc-His6 tag at the C-terminus. The Glu II- cDNA was inserted into the XhoI/EcoRV sites of pBlueBacHis2A (Invitrogen) such that the subunit coding sequence was in frame with the sequence encoding the hexahistidine tag on the N-terminus and the MycHis6 tag from the pcDNA3.1 on the C-terminus.
The EcoRV site at 5' end and SpeI site at 3' end were generated by PCR, using primers 5' CGGATATCGGGATGCTGCTGCTGCTG-3' and 5' CCACTAGTTACAGCTCGTCATGGTCC-3', respectively, from the cDNA clone of mouse Glu II-ß. The cDNA was ligated into pCRII vector from TA cloning kit (Invitrogen) and then was inserted in-frame into the EcoRV/BamHI sites of the mammalian vector, pcDNA3.1B(-)MycHis (Invitrogen) to obtain the vector Glu II-ß-pcDNA containing full-length mouse Glu II-ß cDNA. The Glu II-ß cDNA was inserted into XhoI/KpnI sites of pBlueBacHis2C such that the ß subunit coding sequence was in frame with the sequence encoding the hexahistidine tag on its N-terminus.
Both the transfer plasmids and linearized AcNPV viral DNA (Invitrogen) were cotransfected into Spodoptera frugiperda (Sf9) cells by Insectin-Plus Insect Cell-Specific Liposomes (Invitrogen) according to the manufacturer's instructions.
Recombinant viral plaques were identified by color screening with the presence of 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-gal). Viral DNA prepared from several putative recombinant plaques was analyzed by PCR to confirm the presence of the inserted gene. The positive and purified viruses were amplified by infection of Sf9 cells until the titer reached 1 x 108 pfu (plaque forming unit)/ml.
Insect cell culture
Sf9 cells were grown at 27°C in Grace's insect cell medium (Gibco, Rockville, MD) containing 10% fetal bovine serum in 150-cm2 flasks or 250-ml shaker flasks, rotated at 100 rpm. The titer virus stocks were stored at 4°C and protected from light to ensure maintenance of titer.
Bacoluvirus expression and purification of (His6)-Glu II fusion protein by Ni-NTA-agarose affinity chromatography
Monolayers of Sf9 cells (3 x 108 cells) were coinfected with recombinant baculoviruses encoding Glu II- and Glu II-ß (in 1:1 ratio) at a total multiplicity of 10 and grown for 84 h. The medium was removed by aspiration; the cells were washed with ice-cold phosphate buffered saline, scraped into 25 ml lysis buffer (50 mM phosphate, pH 6.8, 300 mM NaCl, 10 mM imidazole, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM pheylmethylsulfonyl fluoride, and 10% glycerol), and lysed by brief sonication on ice. The lysate was centrifuged at 10,000 x g for 20 min at 4°C to collect the supernatant. The supernatant was incubated with 2 ml of Ni-NTA-agarose slurry (Qiagen) at 4°C for 2 h with gentle agitation. The mixture was placed in a column, and the resin was washed with 20 column volumes of lysis buffer, 80 volumes of wash buffer (containing 50 mM phosphate, pH 7.4, and 300 mM NaCl) with 25 mM imidazole, and 80 volumes of wash buffer with 50 mM imidazole. Bound protein was eluted in four 1-ml fractions with a buffer containing 50 mM phosphate, pH 7.4, 300 mM NaCl, 240 mM imidazole, and 10% glycerol.
Assay of Glu I and II
Enzyme activities were measured essentially as before (Saxena et al., 1987; Shailubhai et al., 1987). Either [Glc-3H]Glc3Man9GlcNAc2 or [Glc-3H]Glc2Man9GlcNAc2 oligosaccharide (7000 cpm) was dissolved in 50 mM phosphate, pH 6.5, and incubated with 20 µl protein sample in 100 µl final volume at 37°C for 30 min. Released [3H]Glc was recovered by elution from Con A–Sepharose 4B and measured by scintillation counting. One unit of enzyme activity is defined as the amount of enzyme protein required to consume 10% of the corresponding substrate in 30 min at 37°C.
Endo H digestion
The affinity-purified Glu II was denatured in 0.1% SDS and 10 mM ß-mercaptoethanol buffer by boiling for 5 min. The sample was digested with 0.04 U Endo H in 200 µl 50 mM citrate buffer, pH 5.5, for 16 h at 37°C.
Binding on Con A–Sepharose 4B
The enzyme sample was suspended in Con A–Sepharose binding buffer (0.2 M sodium acetate, pH 6.8, 1 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2) in a total volume of 200 µl and mixed with the equal volume of Con A–Sepharose 4B resin. The mixture was placed in a column, and the flow through was collected.
Site-specific mutagenesis
Site-specific mutagenesis of Glu II- cDNA was carried out by using the Quick-Change Site Directed Mutagenesis System and the protocol provided by the manufacturer (Stratagene, La Jolla, CA).
Complementary primers containing the desired mutations, flanked by unmodified nucleotide sequences were used in the PCR reaction. The following oligonucleotides and the complementary primers (not shown) were used to generate mutants. The modified codons are underscored, and the mutated amino acid is identified in parentheses: 5'-CTTTATGTTTGGAATAACATGAATGAACCGTCTGTGTTC-3' (D564N), 5'-CTTTATGTTTGGAATGAGATGAATGAACCGTCTGTGTTC-3' (D564E), 5'-CTTTATGTTTGGAATGACATGAATCAACCGTCTGTGTTC-3' (E567Q), 5'-CTTTATGTTTGGAATGACATGAATGACCCGTCTGTGTTC-3' (E567D), 5'-CTTTATGTTTGGAATGACGCGGCTGAACCGTCTGTGTTC-3' (DM565AN566AE), 5'-CTTTATGTTTGGAATGATATGAATGACCCGTCTGTGTTC-3' (D564EMNE567D, double switch), 5'-CTTTATGTTTGGAATGATGCGGCTGACCCGTCTGTGTTC-3' (D564EM565AN566AE567D), 5'-GCTCCTAATCTTGAAGATTACATGTGGAATGTTAATCCGTCTGTGTTC-3' (scramble), and 5'-GACATGAATGAACCGTCTGTGGTCAATGGTCCTGAG-3' (F571A). All mutations were verified by DNA sequencing.
Western blot analysis
Western blots and 10% SDS–PAGE were performed as per standard procedures (Sambrook et al., 1989). Anti-His6 antibody was incubated for 2 h at room temperature at a dilution of 1:1000 in 3% skim milk in Tris-buffered saline (20 mM Tris, pH 7.6, 150 mM NaCl). Both anti-Glu II- and anti-Glu II-ß antibodies were incubated for 2 h at room temperature at a dilution of 1:200 in 3% skim milk in Tris-buffered saline. The blots were developed with the ECL detection system (Amersham Biosciences, Piscataway, NJ).
Analyses of mouse mammary glands at different stages of development
Tissue from a pool of 20–30 mouse mammary glands were at the following stages of development: V, virgin; P7, 7th day of gestation; P14, 14th day of gestation; L1, 24 h postpartum; L7, 7th day of lactation; L11, 11th day of lactation; L14, 14th day of lactation; L21, 21st day of lactation; I2, 2nd day after weaning; I7, 7th day after weaning. This pool was used to prepare microsome membranes as described (Vijay and Oka, 1986). The crude enzyme extracts served as a source of protein and enzyme activity determinations. An aliquot of the total homogenate was saved for DNA estimation (Labarca and Paigen, 1980).
RNA isolation and quantitative RT-PCR
Mouse mammary glands were minced and homogenized with Trizol reagent (Invitrogen), and total RNA was isolated according to the instructions of the manufacturer. Aliquots of 5 µg total RNA were treated with DNase I (Promega, Madison, WI) at 37°C for 15 min to eliminate the contaminating genomic DNA before the RT reaction. The first-strand cDNAs were synthesized with oligo(dT)20 primer (10 µM) and 5 U of SuperScriptII reverse transcriptase (Invitrogen).
Initial experiments optimized the conditions for obtaining specific PCR products in amounts linearly related to the quantity of starting material. The PCR amplification was performed by 27 cycles of 30 s at 94°C, 1 min at 55°C (Glu I, Glu II-, and Glu II-ß) or 65°C (ß-actin), and 2 min at 72°C. The reaction contained 1x PCR buffer, 250 µM each of dNTP, 1.5 mM MgCl2, 0.5 µM forward and reverse primers (Table III), 2 µl of the first-strand cDNA reaction mixture, 2.5 U Taq polymerase (Promega), and 0.5 µl [-32P]dCTP (10 mCi/ml) in a final volume of 50 µl. After amplification, 10 µl of PCR products were separated by eletrophoresis on a 1.5% low-melting agarose gel and identified by ethidium bromide staining. The authenticity of the products was confirmed by DNA sequencing of the fragment after insertion into the pCRII vector from the TA-cloning kit (Invitrogen).
For quantitation, the bands corresponding to the PCR products were excised and the radioactivity counted.
Statistical analysis
Data are expressed as means ± SEM from various numbers of different preparations. The statistical significance of difference between mean values in different groups was evaluated using Student's t-tests.
|
Acknowledgements |
|---|
We are grateful to Dr. Hanne L. Ostergaard for the gifts of cDNA clones of
and ß subunits used in this study. We acknowledge the assistance of Anna Silva and Karen Saunders for preparation of the radioactive oligosaccharide substrates. This research was supported by a grant from the National Institutes of Health (GM59943). |
Footnotes |
|---|
1 To whom correspondence should be addressed; e-mail: ivijay{at}umd.edu
|
Abbreviations |
|---|
Con A, Conacanavalin A; DNM, 1-deoxynojirimycin; Endo H, endoglycosidase H; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RER, rough endoplasmic reticulum; RT, reverse transcription; SDS, sodium dodecyl sulfate
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Arendt, C.W. and Ostergaard, H.L. (1997) Identification of the CD45-associated 116-kDa and 80-kDa proteins as the alpha- and beta-subunits of alpha-glucosidase II. J. Biol. Chem., 272(20), 13117–13125.
D'Alessio, C., Fernandez, F., Trombetta, E.S., and Parodi, A.J. (1999) Genetic evidence for the heterodimeric structure of glucosidase II. The effect of disrupting the subunit-encoding genes on glycoprotein folding. J. Biol. Chem., 274(36), 25899–25905.
Ellgaard, L. and Helenius, A. (2001) ER quality control: towards an understanding at the molecular level. Curr. Opin. Cell Biol., 13(4), 431–437.[CrossRef][Web of Science][Medline]
Flura, T., Brada, D., Ziak, M., and Roth, J. (1997) Expression of a cDNA encoding the glucose trimming enzyme glucosidase II in CHO cells and molecular characterization of the enzyme deficiency in a mutant mouse lymphoma cell line. Glycobiology, 7(5), 617–624.
Frandsen, T.P. and Svensson, B. (1998) Plan -glucosidases of the glycoside hydrolase family 31. Molecular properties, substrate specificity, reaction mechanism, and comparison with family members of different origin. Plant Mol. Biol., 37, 1–13.[CrossRef][Web of Science][Medline]
Heleniuis, A. and Aebi, M. (2001) Intracellular functions of N-linked glycans. Science, 291, 2364–2369.
Henrissat, B. and Davies, G. (1997) Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol., 7, 637–644.[CrossRef][Web of Science][Medline]
Henrissat, B. and Romeu, A. (1995) Families, superfamilies and subfamilies of glycosyl hydrolases. Biochem. J., 311, 350–351.
Hermans, M.M.P., Kroos, M.A., van Beeumen, J., Oostra, B.A., and Reuser, A.J.J. (1991) Human lysosomal glucosidase. Characterization of the catalytic site. J. Biol. Chem., 266, 13507–13512.
Iwanami, S., Matsui, H., Kimura, A., Ito, H., Mori, H., Honma, M., and Chiba, S. (1995) Chemical modification and amino acid sequence of active site in sugar beet -glucosidase. Biosci. Biotechnol. Biochem., 59, 459–463.[Medline]
Keenan, T.W. (2001) Milk lipid globules and their surrounding membranes: a brief history and perspectives for future research. J. Mammary Gland Biol. Neoplasia, 6, 365–371.[CrossRef][Web of Science][Medline]
Kimura, A., Takata, M., Fukushi, Y., Mori, H., Natsui, H., and Chiba, S. (1997) A catalytic amino acid and primary structure of active site in Aspergillus niger -glucosidase. Biosci. Biotechnol. Biochem., 61, 1091–1098.[Medline]
Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagines-linked oligosaccharides. Anuu. Rev. Biochem., 54, 631–664.
Labarca, C. and Paigen, K. (1980) A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem., 102, 344–352.[CrossRef][Web of Science][Medline]
Lee, S.S., He, S., and Withers, S.G. (2001) Identification of the catalytic nucleophile of the Family 31 alpha-glucosidase from Aspergillus niger via trapping of a 5-fluoroglycosyl-enzyme intermediate. Biochem. J., 359(2), 381–386.[CrossRef][Web of Science][Medline]
Lucocq, J.M., Brada, D., and Roth, J. (1986) Immunolocalization of the oligosaccharide trimming enzyme glucosidase II. J. Cell Biol., 102(6), 2137–2146.
Mather, I.H. and Keenan, T.W. (1998) Origin and secretion of milk lipids. J. Mammary Gland Biol. Neoplasia, 3, 275–286.[CrossRef][Web of Science][Medline]
Moremen, K.W., Trimble, R.B., and Herscovics, A. (1994) Glycosidases of the asparagines-linked oligosaccharide processing pathway. Glycobiology, 4, 113–125.
Neville, M.C., Morton, J., and Umemura, S. (2001) Lactogenesis. The transition from pregnancy to lactation. Prediatr. Clin. North Am., 48, 35–52.
Pelletier, M.F., Marcil, A., Sevigny, G., Jakob, C.A., Tessier, D.C., Chevet, E., Menard, R., Bergeron, J.J., and Thomas, D.Y. (2000) The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo. Glycobiology, 10(8), 815–827.
Quaroni, A. and Semenza, G. (1976) Partial amino acid sequence around the essential carboxylate in the active sites of the intestinal sucrose-isomaltase. J. Biol. Chem., 251, 3250–3253.
Rajput, B., Muniappa, N., and Vijay, I.K. (1994) Developmental and hormonal regulation of UDP-GlcNAc:dolichol phosphate GlcNAc-1-P transferase in mouse mammary gland. J. Biol. Chem., 269, 1–8.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 18.47–18.75.
Saxena, S., Shailubhai, K., Dong-Yu, B., and Vijay, I.K. (1987) Purification and characterization of glucosidase II involved in N-linked glycoprotein processing in bovine mammary gland. Biochem. J., 247(3), 563–570.[Web of Science][Medline]
Schmidt, B.F., Ashizawa, E., Jarnagin A.S., Lynn, S., Noto, G., Woodhouse, L., Estell, D.A., and Lad, P. (1994) Identification of two aspartates and a glutamate essential for the activity of endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus. Arch Biochem Biophys., 311(2), 350–353.[CrossRef][Web of Science][Medline]
Shailubhai, K., Pratta, M.A., and Vijay, I.K. (1987) Purification and characterization of glucosidase I involved in N-linked glycoprotein processing in bovine mammary gland. Biochem. J., 247(3), 555–562.[Web of Science][Medline]
Shailubhai, K., Saxena, E.S., Balapures, A.K., and Vijay, I.K. (1990) Developmental regulation of glucosidase I, an enzyme involved in the processing of asparagines-linked glycoproteins in rat mammary gland. J. Biol. Chem., 265, 9701–9706.
Trombetta, E.S., Simons, J.F., and Helenius A. (1996) Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit. J. Biol. Chem., 271(44), 27509–27516.
Trombetta E.S., Fleming K.G., and Helenius, A. (2001) Quaternary and domain structure of glycoprotein processing glucosidase II. Biochemistry, 40(35), 10717–10722.[CrossRef][Medline]
Vijay, I.K. and Oka, T. (1986) Developmental regulation of glycosyltransferases involved in biosynthesis of asparagines-linked glycoproteins in mouse mammary gland. Eur. J. Biochem., 154, 57–62.[Web of Science][Medline]
Watanabe, T., Kobori, K., Miyashita, K., Fujii, T., Sakai, H., Uchida, M., and Tanaka, H. (1993) Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J. Biol. Chem., 268(25), 18567–18572.
Withers, S.G. and Aebersold, R. (1995) Approaches to labeling and identification of active site residues in glycosidases. Protein Sci., 4, 361–372.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Zvonic, M. Lefevre, G. Kilroy, Z. E. Floyd, J. P. DeLany, I. Kheterpal, A. Gravois, R. Dow, A. White, X. Wu, et al. Secretome of Primary Cultures of Human Adipose-derived Stem Cells: Modulation of Serpins by Adipogenesis Mol. Cell. Proteomics, January 1, 2007; 6(1): 18 - 28. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||