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J. Biol. Chem., Vol. 280, Issue 33, 29837-29848, August 19, 2005
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-Mannosidases Involved in N-Glycan Processing and Endoplasmic Reticulum Quality Control*
From the Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602
Received for publication, May 10, 2005 , and in revised form, May 23, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mannosidase I and subsequent recognition by the ER degradation-enhancing -mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The kinetics and energetics of substrate binding and catalysis by members of this family were investigated here by the analysis of wild type and mutant forms of human ER -mannosidase I. The contributions of several amino acid residues and an enzyme-associated Ca2+ ion to substrate binding and catalysis were demonstrated by a combination of surface plasmon resonance and enzyme kinetic analyses. One mutant, E330Q, shown previously to alter general acid function within the catalytic site, resulted in an enzyme that possessed increased glycan binding affinity but compromised glycan hydrolysis. This mutant protein was used in a series of glycan binding studies with a library of mannose-containing ligands to examine the energetics of Man9GlcNAc2 substrate interactions. These studies provide a framework for understanding the nature of the unusual substrate interactions within the family 47 mannosidases involved in glycan maturation and ER-associated glycoprotein degradation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mannosidase I (ERManI) to generate a unique Man8GlcNAc2 isomer product (Fig. 1E) is the key rate-limiting initiation signal (9, 10) that ultimately leads to retrotranslocation of the polypeptide back into the cytoplasm for degradation by the proteasome in a process known as ER-associated degradation (ERAD) (11). Inhibition of ERManI can cause the accumulation of misfolded model glycoproteins in the ER lumen (12-20), and ERManI overexpression has been shown to accelerate the "disposal clock," hastening the disposal of misfolded proteins and even early folding intermediates of wild type proteins (9). Thus, the efficiency of creating fully folded glycoproteins for transport from the ER is defined by a competition between the kinetics of conformational maturation versus the rate of acquiring the key glycan signal for glycoprotein disposal.
Many loss-of-function human genetic diseases result from delayed folding kinetics of potentially functional polypeptides, such as the 
Phe508 mutant of cystic fibrosis transmembrane regulator (21), rather than generating terminally misfolded protein structures (22). Thus, treatment of many protein misfolding disorders could be achieved if pharmacological inhibition of the rate-determining steps for ERAD allowed sufficient time for completion of the protein folding process (7, 23).
ERManI is a member of a larger family of proteins, termed Class 1 mannosidases (24) (CAZy family 47 glycosylhydrolases (25-28)), involved in glycoprotein maturation and disposal. Two other subgroups within this family include a subfamily of hydrolases in the Golgi complex and a subfamily of lectins in the ER. The Golgi -1,2-mannosidases (termed IA (29), IB (30), and IC (31)) are essential for trimming high mannose N-glycans to the Man5GlcNAc2-Asn intermediate necessary for maturation into complex type structures on cell surface and secreted glycoproteins. In the ER, the EDEM subgroups of proteins apparently have no hydrolase activity but act as lectins as a part of the ERAD disposal machinery (2, 32-38). The present models envisage recognition of the glycan structures by the EDEM proteins in a mode similar to substrate recognition during catalysis by the true hydrolases, followed by transfer to the Sec61 translocon pore, retrotranslocation into the cytosol, and proteasomal degradation (2). Thus, understanding how this family of enzymes and lectins accomplish their functions in recognition and catalysis will provide insights into the rate-limiting decisions between glycoprotein maturation and disposal in the secretory pathway.
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-mannosidases have been studied extensively with regard to enzyme kinetics, substrate specificity, structure, and mechanism (24, 39-46). The catalytic domains of enzymes from fungal and mammalian sources have similar ()7 barrel structures (41-45) that are plugged at one end by a 
-hairpin, whereas the opposite end is composed of a broad cleft leading to the catalytic residues in the barrel core (Fig. 1, A and B). In all of the enzymes, a Ca2+ ion is bound at the apex of the -hairpin in the core of the barrel where it is involved in direct interactions with two of the glycone hydroxyls during the catalytic cycle (Fig. 1, A-D) (42, 46). Co-complex structures have been examined between ERManI and the glycone mimic inhibitors, 1-deoxymannojirimycin (dMNJ) and kifunensine (Kif) (Fig. 1F) (42), as well as a co-complex with an uncleaved thiodisaccharide pseudosubstrate (46), revealing a novel conformational itinerary during glycoside bond hydrolysis. Our recent studies suggest that the enzyme binds to the glycone residue in the -1 subsite in a high free energy 3S1 conformation (46), which allows the formation of a ring-flattened 3H4 transition state by a least motion conformational twist of the predisposed sugar ring, and produces an inverted enzymatic product in a 1C4 conformation. Novel general base (Glu599) and general acid (Glu330 and Arg334 acting in a through-water protonation scheme) functions were identified through a combination of kinetic analyses and structure determination of the ERManI-thiodisaccharide co-complex (Fig. 1, C and D) (46). All of the known Class 1 mannosidase structures are essentially identical within the -1 and +1 subsites, suggesting that the catalytic mechanism for bond hydrolysis is conserved among all of the true hydrolases.
Although all of the Class 1 mannosidases cleave Man-1,2-Man linkages, there are significant differences in branch specificities among the different family members (24). ERManI cleaves a single residue from the central branch of the Man9GlcNAc2 substrate to produce a single Man8GlcNAc2 isomer product (Fig. 1E) (39, 40, 47). In contrast, the Golgi subfamily of enzymes recognizes the other terminal Man-1,2-Man branches but instead cleaves the central branch with poor efficiency (48, 49). Thus, these enzymes have a mutually exclusive but complementary specificity for the complete cleavage of Man-1,2-Man linkages on high mannose glycans (41). Putative glycan enzymatic product co-complexes have been isolated for members of both the ER (43) and Golgi (41) subclasses of enzymes, demonstrating that differences in the cleft structures leading from the catalytic core residues confer unique glycan branch specificities for the different subfamily members.
In the studies described here, we have complemented and extended our recent work on the characterization of the ERManI catalytic mechanism (46) by examining for the first time the energetics of substrate binding and catalysis by a class 1 -mannosidase. Kinetic and binding analyses have allowed us to dissect the energetic contributions of individual amino acid residues and the protein-bound Ca2+ ion during substrate binding and catalysis. Through the use of a general acid catalytic mutant that is compromised in hydrolysis, yet maintains substrate binding with similar characteristics to the wild type enzyme, we also mapped the contributions of individual residues in the Man9GlcNAc2 substrate for their interactions with the extended enzyme glycan binding pocket. These studies revealed unanticipated roles for glycan interactions in the 
+1 subsites for facilitating catalysis and substrate specificity. The experimental strategy for the binding and kinetic analyses also provides a framework for further studies on the structural and energetic basis of substrate branch recognition and catalysis by other members of the class 1 (glycosylhydrolase family 47) -mannosidases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A vector (Invitrogen) was used to perform site-directed mutagenesis using the QuikChangeTM mutagenesis kit from Stratagene (La Jolla, CA). Plasmid constructs were then used to transform the Pichia pastoris strain X-33, and Zeocin-resistant colonies were screened for ERManI expression by performing Western blots using conditioned medium from induced cultures as described previously (42, 46). Mutant enzymes were expressed in 1-liter shake flask cultures by induction in BMMY media, and the enzyme was purified from the conditioned media as described previously (42, 46). Glycopeptide and Glycan Isolation—Man9GlcNAc2-glycopeptides were isolated from crude soybean agglutinin by reduction, carboxyamidomethylation, elastase digestion, affinity chromatography using concanavalin A-Sepharose, and further purification by HPLC on a Cosmosil C18 column as described previously (46). Man9GlcNAc2 was liberated from the peptide by peptide:N-glycosidase F digestion (50) and derivatized with pyridylamine (Man9GlcNAc2-PA) (48). Man8GlcNAc2-PA isomers, Man6GlcNAc2-PA, and Man5GlcNAc2-PA, were generated by digestion with either ERManI or Golgi ManIA and isolation by reverse phase HPLC as described (48).
Enzyme, Protein, and Carbohydrate Assays—The purified wild type and mutant enzymes were assayed for 1,2-mannosidase activity using Man9GlcNAc2-PA as substrate as described previously (46). Briefly, the enzyme reactions (20 µl) were carried out in 96-well plates at 37 °C for the indicated times, stopped by the addition of 20 µl of 1.25 M Tris-HCl, pH 7.6, and resolved and quantitated using a Hypersil APS-2 NH2-HPLC column (48). One unit of enzyme activity is defined as the amount of enzyme that generates 1 µmol of Man8GlcNAc2 from Man9GlcNAc2 in 1 min at 37 °C. Protein concentration was determined using the BCA protein assay reagent (Pierce) as described by the manufacturer. Oligosaccharide concentrations were determined by phenol-sulfuric acid assays (51).
Kinetic Analysis—Initial rates (v) for the enzymes were determined at various substrate concentrations ranging from 10 to 300 µM. The catalytic coefficient (kcat) and Michaelis constant (Km) values were determined by fitting initial rates to a Michaelis-Menten function by nonlinear regression analysis using SigmaPlot (Jandel Scientific, San Rafael, CA). kcat/Km values were derived from reciprocal plots of v and [S] where needed. In studies on the temperature dependence of catalysis, values for kcat were obtained between 5 and 40 °C at 5 °C intervals and were used to calculate activation energies (Ea) from the slopes (-Ea/R) of Arrhenius plots (ln(kcat) as a function of 1/T). The thermodynamic activation parameters were described by the Equations 1-3 (52, 53):
 | (Eq. 1) |
 | (Eq. 2) |
 | (Eq. 3) |
where R is the gas constant (8.314 J·mol-1 K-1); kB is the Boltzmann constant (1.3805 x10-23 J·K-1), and h is the Planck constant (6.6256 x 10-34 J·s).
Calcium Equilibrium Analysis—Purified wild type ERManI or the T688A mutant was incubated with 200 mM EGTA for 2 h at 4 °C prior to desalting over a Sepharose G-25 column (1 x 40 cm), which was pretreated with 0.5 M EGTA, followed by pre-equilibration in calcium-free buffer (20 mM MES, pH 7.0, 150 mM NaCl). The calcium-free protein solution was concentrated to 1 mg/ml using an Amicon YM-10 membrane. The calcium content of the protein solution, confirmed by inductively coupled plasma mass spectrometry (ICP-MS), was below 50 ppb.
The total amount of calcium chloride required to generate defined concentrations of free Ca2+ ion (0-500 µM) in an EGTA-containing buffer (20 mM MES, pH 7.0, 150 mM NaCl, 5 mM EGTA) was calculated using WEBMAXCLITE version 1.15 (54) (available online at www.stanford.edu/~cpatton/maxc.html). Solutions of defined Ca2+ ion concentration were prepared, and the pH of the mixture was monitored and adjusted to pH 7.0 by addition of NaOH. All stock solutions were prepared in EDTA-treated plasticware. Total calcium ion concentrations in each solution were confirmed by atomic absorption and used to recalculate the free calcium ion concentration under the conditions of analysis (55).
The calcium-free enzyme solution was diluted in calcium-free buffer to obtain a stock solution of 80 µg/ml for wild type ERManI and 140 µg/ml for the T688A mutant. Aliquots of the enzyme solutions (5 µl) were added to each of the buffers (10 µl) containing defined Ca2+ ion concentrations before addition of 5 µl of 80 µM Man9GlcNAc2-PA. Enzyme reactions were allowed to proceed at 37 °C for 1 h, stopped, and analyzed by NH2-HPLC chromatography as described above. Plotting of mannosidase enzyme activity versus Ca2+ ion concentration revealed a sigmoidal curve similar to data expected for a common equilibrium dialysis experiment, allowing the calculation of the Ca2+ affinity constant (KCa) by nonlinear regression analysis using Equation 4 (55),
 | (Eq. 4) |
where y is equal to the moles of Ca2+ bound per mol of enzyme, measured as units of -mannosidase enzyme activity resulting from Ca2+ binding to the enzyme, and n is the apparent ERManI-specific activity.
Binding Studies by Surface Plasmon Resonance (SPR)—SPR analyses were conducted using a Biacore 3000 apparatus (Biacore AB) with recombinant ERManI immobilized on the SPR chip surfaces at 25 °C by the amine-coupling method as described previously (46). Mock-derivatized flow cells served as reference surfaces. The binding analyses were routinely performed at 10 °C with continuous flow (30 µl/min) of running buffer except in the temperature-dependent interaction studies, which were performed between 5 and 35 °C at 5 °C intervals, consecutively, in an automated method. The running buffer was 10 mM MES, pH 7.0, 300 mM NaCl, and 5 mM CaCl2 in all cases, except in calcium-dependent binding studies. For the latter studies, the chip surface was subjected to overnight treatment with 20 mM MES/NaOH, pH 7.0, 300 mM NaCl, 5 mM EGTA, at a flow rate of 5 µl/min, prior to binding analyses using 20 mM MES/NaOH, pH 7.0, 300 mM NaCl, and 200 µM EGTA as running buffer. Analytes were prepared in the respective running buffers by 2-fold serial dilution to obtain an appropriate concentration range. The binding of Man9-5GlcNAc2-PA and Man9GlcNAc2-glycopeptide glycans was analyzed in a concentration series (0.4-400 µM) over a low density immobilization surface (46) of recombinant protein (3000 response units), whereas dMNJ and Kif (2-1000 µM) were analyzed over a high density immobilization surface (46) of recombinant protein (10,000 response units). The base line returned to the original response in 5 min for all analytes described here without a further regeneration procedure, except for analyses using Kif as the analyte, which did not dissociate from the chip surface even with extensive washing.
SPR data for each concentration of analyte were collected in duplicate and globally fit to a 1:1 Langmuir binding algorithm model to calculate the on-rate (ka), the off-rate (kd), and the equilibrium dissociation constant (kd/ka = KD) using the BIAevaluation 3.1 software (56). Alternatively, the maximal equilibrium sensorgram values were used to plot a saturation binding curve and calculate values for the equilibrium dissociation constant (KD) directly.
KD values measured at different temperatures were used to calculate thermodynamic parameters (56, 57) of binding using van't Hoff equation (Equation 5), (58) and Gibbs free energy change for binding (G) was also calculated (Equation 6). The van't Hoff equation allows the calculation of thermodynamic parameters using the linear relationship of y = ln(KD) versus x = 1/T, which gives a slope of H/R and an intercept of -S/R (58).
 | (Eq. 5) |
 | (Eq. 6) |
The effects of temperature on the association rates (ka) and dissociation rates (kd) were independently determined using the Eyring equation (Equation 7) (58).
 | (Eq. 7) |
Similar to the van't Hoff analysis, the Eyring equation allows thermodynamic parameters to be determined from measured ka and kd values at different temperatures by a linear relationship of y = Rln(hka/kBT) or y = Rln(hkd/kBT) versus x = 1/T, where the slope and the intercept of the Eyring plots are -H
and S, respectively (58).
Isothermal Titration Calorimetry (ITC)—Calorimetry measurements were performed with a 4200-ITC calorimeter (Calorimetry Sciences Corp., Lindon, UT) as described (59, 60). Protein solutions for ITC analysis were dialyzed overnight against buffer containing 20 mM MES, pH 7.0, 150 mM NaCl, 5 mM CaCl2 and 0.75 M 3-(1-pyridino)-1-propane sulfonate (NDSB201; Calbiochem) at 4 °C. The ligand solutions of Kif and dMNJ were prepared by diluting the compounds in the buffer used for protein dialysis. Aliquots (5-10 µl) of the ligand solution (1-5 mM) were automatically delivered into 1.3 ml of protein solution (1-2 mg/ml) in the reaction cell. The calorimetry cell was allowed to return to equilibrium for 4 min prior to the next injection. The data analysis was performed using DataWork and BindWork software provided by manufacturer. Protein and glycan molecular structure figures were prepared using MacPymol (version 0.95)2 to generate rasterized images.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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+1 residues (Arg461), and one residue that interacts with both the -1 and +2 subsite residues (Arg597) (Fig. 1, C and D), and characterized their roles in catalysis and substrate binding. Phe659 was shown previously to provide van der Waals interactions to the C4-C5-C6 region of the glycone in the -1 subsite during catalysis (Fig. 1D) (42, 46). Thr688 is positioned at the apex of the -hairpin in the core of the ()7 barrel where it has been shown to be the sole protein residue that directly coordinates the bound Ca2+ ion through both its O-
and carbonyl oxygens (Fig. 1D) (42, 46). The other four points of coordination of the Ca2+ ion to the enzyme are indirect through-water interactions with carboxylate side chains in the core of the barrel. Arg461 interacts with several residues in the core of the Man9GlcNAc2 substrate (Fig. 1, D and E, residues M7, M4, and M3) and has been proposed to contribute to branch specificity for Saccharomyces cerevisiae ERManI (61). A Leu residue is found at the equivalent position in the Golgi subclass of enzymes. Previous mutagenesis studies generating the equivalent of an R461L mutant for S. cerevisiae ERManI (61) resulted in an enzyme that had a hybrid activity between the specificity of ERManI that cleaves only the central branch mannose residue and the Golgi mannosidases that cleave the remaining 1,2-Man residues down to Man5GlcNAc2. Finally, Arg597 appears to play a dual role in hydrogen bonding to the O-6' hydroxyl oxygen of the glycone in the -1 subsite via NH1 and the O-4' hydroxyl of the mannose in the +2 subsite via NH2. Each of the mutants (T688A, F659A, R461A, R461L, and R597A) was expressed in P. pastoris as a secreted catalytic domain, and the detailed enzyme kinetic parameters for hydrolysis of Man9GlcNAc2-PA were determined and are summarized in Table I. The pH optima for all of the mutant enzymes were slightly below the value for wild type ERManI (pH 6.5-6.8 versus 7.0 for wild type; Table I). The catalytic rates (kcat) and the catalytic efficiencies (kcat/Km) of Man9GlcNAc2 cleavage for all of the mutants were significantly decreased, resulting in a range of kcat/Km values that varied from 0.6 to 12% of wild type values. Surprisingly, the Km value for the T688A mutant was reduced 7.3-fold, whereas the Phe659 mutant remained unaffected. In contrast, the Km values for the other mutants were all significantly increased by 3-5-fold.
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1,2-mannoside residues from Man9GlcNAc2-PA to Man8-6GlcNAc2-PA, as described previously for an equivalent mutant of S. cerevisiae ERManI (61) (data not shown). The 53-fold decrease in kcat and 3-fold increase in Km (Table I) indicated that although the amino acid substitution relaxed the specificity of the enzyme for glycan cleavage beyond Man9GlcNAc2, the enzyme lost significant catalytic efficiency as a result of the mutation. In contrast, the R461A or R597A mutants were unable to cleave beyond Man8GlcNAc2. However, the catalytic rate of the R461A mutant was intermediate between the wild type enzyme and the R461L mutant. These data suggest that the removal of the Arg461 side chain in the R461A mutant moderately compromised catalysis while retaining ERManI substrate specificity. In contrast the R461L mutant was altered in substrate specificity, although its catalytic efficiency was severely compromised. The catalytic rates (kcat) of wild type ERManI and the E330Q and T688A mutants were also obtained from initial rates measured at different temperatures but under optimal pH conditions for the respective enzymes (pH 7.1, 5.3, and 6.5 for wild type, E330Q, and T688A, respectively). The enzyme activities increased with temperature, and activation energies (Ea) were calculated from the slopes of the Arrhenius plots (supplemental Fig. 1A and supplemental Table I). The wild type enzyme and T688A mutant appear to have similar trends, with significant enthalpy and entropy contributions to the activation energy, whereas the E330Q mutant had a slightly reduced entropic contribution (supplemental Fig. 1A and supplemental Table I).
Effect of the T688A Mutant of ERManI on Ca2+ Ion Affinity and Enzyme Activity—The role of the protein-bound Ca2+ ion in catalysis by ERManI was determined by depleting wild type ERManI or the T688A mutant of bound Ca2+ and then performing enzyme assays at defined Ca2+ concentrations controlled by the presence of the divalent cation chelator EGTA. In the absence of any added Ca2+, both proteins exhibited no detectable enzyme activity (supplemental Fig. 2). Addition of Ca2+ resulted in a progressive appearance of enzyme activity, allowing the calculation of the Ca2+ affinity for the enzyme, KCa, by curve-fitting. The KCa values for the wild type enzyme (0.24 ± 0.02 µM) and the T688A mutant (0.15 ± 0.01 µM) were quite similar, yet the specific activity of the T688A mutant was generally 
15-fold lower than the wild type enzyme at all Ca2+ concentrations where activity could be detected. The observation that the T688A mutant is compromised in catalysis (reduced kcat) but increased in substrate binding affinity (reduced Km), while being essentially unaltered in Ca2+ binding affinity, indicates that the mutation has a direct effect on catalysis rather than acting through a reduced affinity for binding and coordinating Ca2+. The KCa value for wild type ERManI was similar to the affinity constants previously determined for yeast ERManI (62) and rabbit liver Golgi ManIA (63).
Glycan and Inhibitor Binding Affinity Measurements to Human ERManI—In addition to kinetic analysis, the binding affinities of inhibitors and high mannose oligosaccharides to wild type and mutant forms of ERManI were also examined by SPR. Prior SPR studies with wild type and mutant forms of ERManI revealed significant alterations in the on-rates (ka) and off-rates (kd) of binding to dMNJ and Man9GlcNAc2-glycopeptide ligands (46). Correlations were made with the positions of the mutations and their impacts on ligand binding affinity. In the present study, we performed similar types of SPR studies with wild type ERManI in the presence and absence of Ca2+, as well as testing the effects of the T688A, F659A, R597A, R461L, and R461A mutants on the binding of Man9GlcNAc2-glycopeptide, dMNJ, or Kif ligands (Fig. 2 and Table II). As described previously (46), the equilibrium dissociation constants (KD) could be measured from a combination of the on-rates (ka) and off-rates (kd) determined by curve-fitting of the SPR sensorgrams, where KD = kd/ka. When the on-rates and off-rates were too fast for accurate measurement, plotting of a saturation curve for the equilibrium values of the binding sensorgrams (Fig. 2, inset plots) allowed an alternative means of determining the KD values (46).
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)7 barrel influences the rate of glycone binding to the -1 subsite, but the overall equilibrium binding affinity of the larger Man9GlcNAc2 substrate is not significantly influenced by the absence of the divalent cation.
Similar to the reduced on- and off-rates for Man9GlcNAc2-glycopeptide binding to the Ca2+-depleted enzyme, the T688A mutant also had significantly reduced on- and off-rates for high mannose glycan binding, but an 50-fold increase in the equilibrium binding affinity (Table II) similar to the increase in glycan binding affinity previously observed for a E330Q general acid mutant (46). The increased glycan binding affinity for the T688A mutant, when combined with the significantly reduced kcat (Table I) and an unaltered Ca2+ binding affinity (supplemental Fig. 2), indicates that the reduced catalytic turnover results in a substrate that is stabilized in an uncleaved form in the active site. This altered environment is less favorable for binding to dMNJ (5-fold decrease in binding affinity), which we have proposed previously (42, 46) to resemble the conformation of the enzymatic product.
In contrast to the effects of the T688A mutant at the base of the -1 subsite, mutation of Phe659 (F659A), which provides van der Waals interactions with the -1 subsite residue, had a minimal effect on Man9GlcNAc2 binding (4-fold reduction in KD, see Table II), consistent with the lack of an effect of the mutation on the Km with the same glycan substrate. However, the binding of dMNJ was completely abolished for this mutant enzyme (KD > 10 mM; Fig. 2), consistent with 123-fold reduction in kcat (Table I). These data suggest that Phe659 plays a key role in glycone binding within the -1 subsite to promote catalysis.
Finally, the binding of Kif to wild type ERManI was examined by SPR. The equilibrium binding affinity for this inhibitor could only be estimated at <30 nM, because there was no detectable dissociation of the compound from the enzyme, even after extensive washing (Fig. 2). Inhibitor binding studies by ITC confirmed the tight binding by the inhibitor, which was highly exothermic and had a strongly favorable enthalpy of binding (see supplemental Table II). The E330Q, D463N, and T688A mutants also formed stable nondissociable complexes with Kif (data not shown). To test the role of the van der Waals interactions between Phe659 and the inhibitor at the base of the -1 subsite, we examined the binding of Kif to the F659A mutant. In contrast to the wild type enzyme, binding of Kif to the F659A mutant was reversible (Fig. 2), with measurable on- and off-rates and a KD of 1.45 µM (Table II).
Binding of Man9GlcNAc2 to the R461A and R597A mutants exhibited reduced on- and off-rates, but the KD values were similar to the wild type enzyme (data not shown). In contrast, the R461L mutant exhibited no detectable binding (KD > 1 mM) to Man9GlcNAc2 (data not shown), consistent with the poor catalytic efficiency of this mutant enzyme that has a hybrid activity between ERManI and the Golgi subclass of enzymes.
Temperature and pH Dependence of Glycan Binding—In an effort to examine the temperature dependence of glycan binding to the wild type and mutant forms of ERManI, we performed a series of SPR binding studies with Man9GlcNAc2 as ligand at temperatures between 5 and 35 °C. The sensorgram responses for wild type ERManI, E330Q, and T688A (supplemental Fig. 3) are representative of the effects of temperature on the respective enzymes. Because of the fast on- and off-rates, binding to the wild type enzyme could only be measured at the equilibrium plateau values for the sensorgrams, and below 15 °C there was little effect of increasing temperature (supplemental Fig. 3). At higher temperatures there was a progressive decrease in the sensorgram amplitude, presumably as a result of a combination of binding and hydrolysis of the ligand and subsequent release of the enzymatic product. For the T688A mutant, the off-rate progressively increased with increasing temperature, whereas the equilibrium sensorgram values increased up to 20 °C and then progressively decreased (supplemental Fig. 3). We have interpreted these data to indicate that glycan binding was more prevalent than glycan hydrolysis at low temperature but that increased hydrolysis at higher temperature led to an increased off-rate and an inflection for the equilibrium sensorgram values. In contrast, the off-rate for the E330Q mutant was considerably less influenced by increasing temperature, whereas the on-rate increased with temperature, leading to an increase in equilibrium binding (supplemental Fig. 3). Similar results were revealed when the binding data were subjected to van't Hoff analysis, where the slopes of the plots indicated that the temperature dependence of the KD for the T688A mutant was greater than for the E330Q mutant (supplemental Fig. 1B). Eyring analysis demonstrated that the temperature dependence of the KD for the T688A mutant was mainly due to an increase in the dissociation rate with increasing temperature (supplemental Fig. 1, C and D). Calculation of the thermodynamic parameters for the glycan association and dissociation (supplemental Table I) indicated that there was a significantly greater entropic contribution to glycan dissociation for the T688A mutant by comparison to the E330Q mutant, as would be predicted for the greater temperature dependence of dissociation.
To confirm the temperature dependence of enzyme activity under the conditions used for SPR analysis (pH 7.0) we compared the specific activities of the various enzyme forms at 10 and 37 °C at this pH. The wild type enzyme had a 30-fold increase in specific activity between 10 and 37 °C, whereas the T688A mutant increased 10-fold and the E330Q mutant increased only 2-fold over the same temperature range (data not shown). Because the E330Q mutant has a reduced pH optimum for catalysis (pH 5.3) relative to the wild type enzyme (pH 7.1) (46), we also tested the pH dependence of glycan binding by the E330Q mutant. At pH values 7.0, the E330Q mutant exhibits high affinity binding (KD < 1 µM) (Table III and supplemental Fig. 4), whereas at pH values below 7.0 both the on- and off-rates are altered to result in an enzyme with a 46-fold lower binding affinity at pH 5.0. We interpret the alterations in on- and off-rates and decrease in glycan affinity to reflect an increase in glycan hydrolysis under conditions closer to the pH optimum of the mutant enzyme. These data indicate that the E330Q mutant is an effective model for binding analyses under our conditions for SPR (pH 7.0, 10 °C) because, in contrast to wild type ERManI or the T688A mutant, there are minimal contributions of ligand hydrolysis.
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G values for binding. The contributions of individual residues or groups of residues were subsequently calculated by a combination of difference calculations (G values) using the rationale shown in Fig. 3B and Table V. These calculations indicate that binding of the glycone residue in the -1 subsite contributes only 21% (-1.68 kcal/mol) to the overall binding energy of a Man9GlcNAc2 ligand (Table V and Figs. 3C and 4), whereas the +1 subsite residue (M7 in Figs. 1E and 4) contributes 52% (4.2 kcal/mol) to the overall binding energy. Residues M9, M8, and M11 (Fig. 1E) together contribute 19% (1.53 kcal/mol) to the glycan binding energy, whereas the remainder of the glycan contributes 0.42 kcal/mol (6%), and the peptide backbone contributions are negligible (0.19 kcal/mol) (Fig. 3C and Table V).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-mannosidases play key and diverse roles in glycan maturation and disposal of misfolded glycoproteins in the ER (2, 9, 24, 32-36, 38). Prior studies have revealed that the overall protein fold for the catalytic domain of members of this family is an ()7 barrel structure with a catalytic site in the core of the barrel (41-45). Co-complex structures between ERManI and dMNJ, Kif (42), or a mannobiose thiodisaccharide (46) revealed the conformational itinerary of the glycone during catalysis. Distortion of the glycone into a 3S1 conformation during substrate binding has been proposed to predispose the substrate for hydrolysis by a least motion conformational twist through a ring-flattened 3H4 transition state producing an inverted enzymatic product in a 1C4 conformation (46). Several questions remain regarding the energetics of substrate binding during catalysis and the nature of the active site chemistry. Conservation in the positions of key residues in the -1 and +1 enzyme subsites indicates that both the ER and Golgi subfamilies of hydrolases cleave Man-1,2-Man linkages by a similar mechanism. However, the ER and Golgi enzymes recognize distinctive terminal branches of Man9GlcNAc2 substrates. Prior structural data on putative enzyme-product co-complexes for both the ER and Golgi enzymes support the proposal that the respective enzymes have distinctive geometries in the clefts leading from the barrel cores that engage the > +1 residues of the respective substrates and confer the unique branch specificities for the different enzyme subfamilies (41). In an effort to understand the energetics of substrate binding and catalysis for the class 1 mannosidases, we have examined the interactions between the wild type and mutant forms of ERManI by using various substrates and inhibitors.
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Initial studies of inhibitor binding examined the energetics and kinetics of interactions by ITC and SPR. Both approaches indicated that binding of the inhibitors was highly exothermic, and the KD values from ITC were in close agreement with Ki values from catalytic measurements and binding affinities by SPR (supplemental Table II). The major difference in structure between dMNJ and Kif is the fused five-membered ring in the latter compound (Fig. 1F) that causes Kif to be "pre-loaded" in the high free energy 1C4 conformation prior to binding to the enzyme (46). The restricted conformation and the additional interactions between the enzyme and the Kif five-membered ring were proposed to account for the higher binding affinity of Kif in comparison to dMNJ (42). Surprisingly, binding studies by SPR indicated that the on-rate for binding to wild type ERManI was comparable between dMNJ and Kif, but the off-rate for Kif was considerably slower. Thus, the restricted conformation of the Kif six-membered ring did not accelerate binding of the inhibitor but significantly slowed dissociation. SPR binding studies between Kif and the F659A mutant, which should eliminate the van der Waals interactions with the C4-C5-C6 region of the ring-constrained inhibitor, significantly increased the off-rate and lowered the overall inhibitor binding affinity. Consistent with the lower binding affinity for Kif to the mutant enzyme, binding of dMNJ to the F659A mutant was not even detectable by SPR (KD >10 mM). These data indicate that Phe659 plays a critical role in stabilizing the glycone in the active site and that the van der Waals interactions with the ring-constrained inhibitor contributes to its slow dissociation rate from the active site. By extension, the greater ring flexibility of dMNJ or the substrate/product glycone residue associated with the wild type enzyme would be predicted to contribute an entropic component favoring dissociation from the active site. Consistent with a role for Phe659 in inhibitor binding, the F659A mutant also caused a 123-fold reduction in kcat, suggesting that interactions between Phe659 and the glycone in the -1 subsite play a significant role in constraining the substrate into the 3S1 conformation required for catalysis.
At the base of the -1 subsite a protein-bound Ca2+ ion interacts directly with the glycone 2'- and 3'-hydroxyl residues. The ion is coordinated directly with the O- and carboxyl oxygens of Thr688 and indirectly with four water molecules associated with Glu residues in the core of the -1 subsite. Two types of studies examined the roles of the Ca2+ ion in glycan binding and catalysis. First, SPR binding studies on the Ca2+-depleted enzyme indicated that the ion contributes to glycan on- and off-rates, but the enzyme had an almost identical equilibrium binding affinity for the Man9GlcNAc2 ligand as the enzyme containing bound Ca2+. Binding of dMNJ was drastically reduced, with a predominant effect on reducing the on-rate of the inhibitor. Second, altering the Thr688 side chain to an Ala did not significantly alter Ca2+ binding affinity for the enzyme, but it reduced kcat by 61-fold and increased Man9GlcNAc2 binding affinity by 50-fold. These data suggest that alterations in the -1 subsite can lead to an increased binding affinity for an uncleaved substrate. The data also suggest that glycone interactions with the enzyme-bound Ca2+ ion, in the context of the appropriate tethering by Thr688, directly facilitate catalysis.
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The results of the glycan binding studies revealed that the +1 subsite residue (residue M7) contributes a majority (52%) of the binding energy for a Man9GlcNAc2 ligand, whereas the glycone binding to the -1 subsite contributes only 21% of the binding energy. Another 19% of the binding energy is contributed from the other peripheral 1,2Man residues, and 7.6% of the binding energy comes from core glycan residues. These data are consistent with binding data obtained from the respective ERManI mutants (Fig. 4). Mutations in the -1 subsite or conditions that significantly reduce dMNJ binding affinity or catalysis either have a minimal effect on Man9GlcNAc2 glycan binding (F659A mutant or Ca2+ depletion) or actually increase glycan binding affinity (T688A and E330Q mutants). Thus, the contributions of the +1 subsite residues to the overall binding affinity can compensate for compromised interactions with glycan substrates in the -1 subsite (Fig. 4).
In contrast, alterations in the +1 subsite, such as the D463N or R461L mutants (Fig. 4), abolished glycan binding (KD values >1 mM). The former side chain anchors the interactions between the enzyme and the M7 residue in the +1 subsite by hydrogen bonding with the sugar O-3' and O-4' hydroxyls (46). For the latter residue, Arg461 has been shown to form a matrix of interactions with mannose residues M7, M4, and M3 (43, 61), yet substitution with an Ala residue (R461A) resulted in a near wild type binding affinity for Man9GlcNAc2. In contrast, the R461L mutant has no detectable Man9GlcNAc2 binding, confirming that there is likely a problem with steric hindrance in the latter mutant.
In conclusion, the combined studies on catalysis and glycan binding by wild type and mutant ERManI forms revealed an active site cleft that promotes substrate binding predominantly through interactions with the +1 subsite residues (Fig. 4). How do these data compare with our emerging model for catalysis by class 1 -mannosidases (46)? The docking of the glycone residue in a high free energy 3S1 conformation, predisposed for glycoside bond hydrolysis, is partly facilitated by van der Waals interactions with Phe659 and additional hydrogen bonding to -1 subsite residues. However, the predominant source for substrate binding energy is provided by interactions with the +1 residue, dominated by the pair of hydrogen bonds from Asp463 to the 3'- and 4'-hydroxyls of the +1 subsite residue (Fig. 4). These latter interactions likely offset the entropic penalty for binding to a high free energy glycone conformation in the -1 subsite, creating favorable energetics for the conformational distortion required for glycoside bond hydrolysis. Additional binding energy and branch specificity are provided by the interactions with the > +1 subsite residues. Interactions between the glycone residue and the enzyme-bound Ca2+ ion influence the association and dissociation rates for glycan substrates, but do not significantly increase their respective equilibrium binding affinities. However, the Ca2+ ion does promote catalysis, presumably through the assistance of Thr688, the sole residue involved in direct coordination with the divalent cation. In the absence of the Thr688 side chain, a solvent water molecule likely replaces the lost point of Ca2+ ion coordination. Ca2+ binding affinity is not reduced in the T688A mutant, yet the catalytic rate is significantly reduced, and glycan binding affinity is surprisingly increased. These data strongly suggest that Thr688 aids in Ca2+-mediated catalysis, either through appropriate positioning of the Ca2+ ion in the active site or by providing appropriate electrostatics to the divalent cation. It is worth noting that the water nucleophile in the inverting catalytic mechanism is directly coordinated to the Ca2+ ion (46), and an isosteric amide substitution of the adjoining general base residue (E599Q) reduced catalysis by 13,000-fold (Fig. 4) (46). However, this mutation did not increase the binding affinity for the uncleaved substrate. Thus, altering the electrostatics of the general base function for activation of the water nucleophile does not account for the increased glycan binding affinity for the T688A mutant. An alternative role for the Thr688 side chain could be a mechanical tethering of the Ca2+ ion in a favorable position adjacent to the -1 subsite that is required for efficient catalysis. A similar effect of reduced catalysis and increased binding of an uncleaved substrate is found for the general acid mutant E330Q. Future studies on the structures of co-complexes between the T688A and E330Q mutants with uncleaved substrates or substrate analogs should provide insights into the roles of the glycone conformational changes and the enzyme-associated Ca2+ ion in glycan hydrolysis.
The use of the E330Q mutant in SPR binding studies was also shown to be an effective tool in assessing the binding contributions of respective residues within the Man9GlcNAc2 substrate. Applying a similar approach to map the contributions of oligosaccharide substrate residues for the other Class 1 mannosidases will reveal the molecular basis of substrate recognition and specificity for this diverse enzyme family. More importantly, the ability to measure detailed binding affinities and kinetics using the equivalent of the E330Q mutant as a model should provide critical information for the analysis of new selective inhibitors for class I mannosidases as potential targets for human protein misfolding disorders.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains Tables 1 and 2 and Figs. 1, 2, 3, 4.
To whom correspondence should be addressed: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602. Tel.: 706-542-1705; Fax: 706-542-1759; E-mail: moremen{at}uga.edu.
1 The abbreviations used are: ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; EDEM, ER degradation enhancing -mannosidase-like protein; ERManI, ER -mannosidase I; Golgi ManIA, Golgi -mannosidase IA; dMNJ, 1-deoxymannojirimycin; Kif, kifunensine; HPLC, high performance liquid chromatography; PA, pyridylamine; SPR, surface plasmon resonance; ITC, isothermal titration calorimetry; MES, 4-morpholineethanesulfonic acid.
2 W. L. Delano, Pymol Molecular Graphics System, available online at www.pymol.org.
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