Role of C-terminal region of Staphylococcal nuclease for foldability,stability, and activity

The role of the C-terminal region of Staphylococcal nuclease (SNase) was examined by deletion mutation. Deletions up to eight residues do not affect the structure and function. The structure and enzymatic activity were partially lost by deleting Ser141-Asn149 (Delta141-149), and deletion of Trp140-Asn149 (Delta140-149) resulted in further loss of structure and activity. A 13-residue deletion showed the same effect as the 10-residue deletion. Both Ser141Gln and Ser141Ala mutations for an eight-residue deletion mutant did not alter properties as well as Ser141A1a for full-length SNase. In contrast, Trp140Ala mutation for Delta141-149 shows the same effect as the deletion of Trp140. Trp140Ala mutation for full-length SNase causes the loss of native structure. These observations indicate the significance of the 140th and the 141st residues. The side-chain of the 140th residue is required to be tryptophan; however, the backbone of the 141st residue is solely critical for foldability, but the side-chain information is not crucial. All of the mutants that take a non-native conformation show enzymatic activity and inhibitor-induced folding, suggesting that foldability is required for the activity.     

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of conserved glycine residues and the identification of additional conserved protein motifs in eNTPDases.

Glycine residues are recognized as important structural determinants in nucleotide-binding domains of many enzymes. The functional significance of seven glycine residues invariant in all 22 eNTPDase sequences was therefore examined. Glycine-to-alanine mutants of eNTPDase3 were analyzed for nucleotidase activities and tertiary and quaternary structure changes. Mutations G98A and G183A had modest effects on ATPase and ADPase activities. The G141A mutation resulted in 4- to 5-fold decreased nucleotidase activity, while the G222A mutation decreased ATPase activity 20-fold, and ADPase activity 6-fold. Unlike the other five glycine mutants, the G263A and G462A mutations caused significant loss of nucleotidase activity which was observed concomitant with lower protein expression levels, large-scale changes in tertiary and quaternary protein structure, and decreased trafficking to the plasma membrane. Thus, these data identify glycine residues that are essential for enzymatic activity and the tertiary and quaternary structure of eNTPDase3. Further, two additional conserved regions in the eNTPDases are identified, apyrase conserved regions ACR1a and ACR4a, which may be involved in phosphate binding/hydrolysis and protein folding, respectively.     

Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5–188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560 kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70 kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610 kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. In vitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.     

Two glutamic acids in chitosanase A from Matsuebacter chitosanotabidus 3001 are the catalytically important residues.

Chitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant k(cat) for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)(6) was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.     

Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is approximately 90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.     

Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.     

The fourth transmembrane domain of the Helicobacter pylori Na+/H+ antiporter NhaA faces a water-filled channel required for ion transport

Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.     

Xylanase XYL1p from Scytalidium acidophilum: Site-directed mutagenesis and acidophilic adaptation

The role of residues Asp60, Tyr35 and Glu141 in the pH-dependent activity of xylanase XYL1p from Scytalidium acidophilum was investigated by site-directed mutagenesis. These amino acids are highly conserved among the acidophilic family 11 xylanases and located near the catalytic site. XYL1p and its single mutants D60N, Y35W and E141A and three combined mutants DN/YW, DN/EA and YW/EA were over-expressed in Pichia pastoris and purified. Xylanase activities at different pH’s and temperatures were determined. All mutations increased the pH optimum by 0.5–1.5 pH units. All mutants have lower specific activities except the E141A mutant that exhibited a 50% increase in specific activity at pH 4.0 and had an overall catalytic efficiency higher than the wild-type enzyme. Thermal unfolding experiments show that both the wild-type and E141A mutant proteins have a T_m maximum at pH 3.5, the E141A mutant being slightly less stable than the wild-type enzyme. These mutations confirm the importance of these amino acids in the pH adaptation. Mutant E141A with its enhanced specific activity at pH 4.0 and improved overall catalytic efficiency is of possible interest for biotechnological applications.     

Mutations in Tyr808 reveal a potential auto-inhibitory mechanism of guanylate cyclase-B regulation

In this study, Tyr⁸⁰⁸ in GC-B (guanylate cyclase-B), a receptor of the CNP (C-type natriuretic peptide), has been shown to be a critical regulator of GC-B activity. In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr⁸⁰⁸ markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na₃VO₄ and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na₃VO₄-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr⁸⁰⁸ is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.     

Cover Image,Volume 88, Issue 6

Hsp16.3, a molecular chaperone, plays a vital role in the growth and survival of Mycobacterium tuberculosis inside the host. We previously reported that deletion of three amino acid residues (¹⁴²STN¹⁴⁴) from C-terminal extension (CTE) of Hsp16.3 triggers its structural perturbation and increases its chaperone activity, which reaches its apex upon the deletion of its entire CTE (¹⁴¹RSTN¹⁴⁴). Thus, we hypothesized that Arg141 (R141) and Ser142 (S142) in the CTE of Hsp16.3 possibly hold the key in maintaining its native-like structure and chaperone activity. To test this hypothesis, we generated two deletion mutants in which R141 and S142 were deleted individually (Hsp16.3ΔR141 and Hsp16.3ΔS142) and three substitution mutants in which R141 was replaced by lysine (Hsp16.3R141K), alanine (Hsp16.3R141A), and glutamic acid (Hsp16.3R141E), respectively. Hsp16.3ΔS142 or Hsp16.3R141K mutant has native-like structure and chaperone activity. Deletion of R141 from the CTE (Hsp16.3ΔR141) perturbs the secondary and tertiary structure, lowers the subunit exchange dynamics and decreases the chaperone activity of Hsp16.3. But, the substitution of R141 with alanine (Hsp16.3R141A) or glutamic acid (Hsp16.3R141E) perturbs its secondary and tertiary structure. Surprisingly, such charge tampering of R141 enhances the subunit exchange dynamics and chaperone activity of Hsp16.3. Interestingly, neither the deletion of R141/S142 nor the substitution of R141 with lysine, alanine and glutamic acid affects the oligomeric mass/size of Hsp16.3. Overall, our study suggests that R141 (especially the positive charge on R141) plays a crucial role in maintaining the native-like structure as well as in regulating subunit exchange dynamics and chaperone activity of Hsp16.3.     

Elucidation of information encoded in tryptophan 140 of staphylococcal nuclease

We investigated the role of W140 in the folding of Staphylococcal nuclease. For this purpose, we constructed the 19 possible substitution mutations at residue 140. Only three mutants, W140F, W140H, and W140Y, adopted native-like structures under physiological conditions and showed native-like enzymatic activities. In contrast, the other 16 mutants took on compact unfolded structures under physiological conditions and the enzymatic activities of these mutants were decreased to approximately 70% of wild-type levels. These 16 mutants maintained substrate-induced foldability. These results strongly indicate that the side-chain information encoded by residue 140 is essential to maintain a stable native structure, and that this residue must be an aromatic side chain. The order of thermal stability was wild type > W140H > W140F = W140Y. Therefore, the five-membered nitrogen-containing ring of the indole is thought to bear the essential information. In the crystal structure of staphylococcal nuclease, the five-membered ring is at the local center of the C-terminal cluster through hydrophobic interactions. This cluster plays a key role in the interaction connecting the C-terminal region and the N-terminal beta-core. Mutants other than W140H, W140F, and W140Y lost the ability to form the local core, which caused the loss of the long-range interactions between the C-terminal and N-terminal regions. Inhibitor or substrate binding to these mutants compensates for the lack of long-range interactions generated by W140.     

The importance of C-terminal aspartic acid residue (D141) to the antirestriction activity of the ArdB (R64) protein

Antirestriction proteins of the ArdB/KlcA family are specific inhibitors of restriction (endonuclease) activity of type-I restriction/modification enzymes. The effect of conserved amino acid residues on the antirestriction activity of the ArdB protein encoded by the transmissible R64 (IncI1) plasmid has been investigated. An analysis of the amino acid sequences of ArdB homologues demonstrated the presence of four groups of conserved residues ((1) R16, E32, and W51; (2) Y46 and G48; (3) S81, D83 and E132, and (4) N77, L(I)140, and D141) on the surface of the protein globule. Amino acid residues of the fourth group showed a unique localization pattern with the terminal residue protruding beyond the globule surface. The replacement of two conserved amino acids (D141 and N77) located in the close vicinity of each other on the globule surface showed that the C-terminal D141 is essential for the antirestriction activity of ArdB. The deletion of this residue, as well as replacement by a hydrophobic threonine residue (D141T), completely abolished the antirestriction activity of ArdB. The synonymous replacement of D141 by a glutamic acid residue (D141E) caused an approximately 30-fold decrease of the antirestriction activity of ArdB, and the point mutation N77A caused an approximately 20-fold decrease in activity. The residues D141 and N77 located on the surface of the protein globule are presumably essential for the formation of a contact between ArdB and a currently unknown factor that modulates the activity of type-I restriction/modification enzymes.     

Identifying functional residues in Arabidopsis thaliana zeta class glutathione S-transferase through screening inactive point mutants

The functional residues of z-class glutathione S-transferase were identified by screening inactive point mutants from a random mutagenesis library. First, a random mutant library was constructed using error-prone polymerase chain reaction, and then candidate inactive mutants were screened by a high-throughput colorimetric assay. Twenty-five mutants were obtained, and 12 that formed inclusion bodies were discarded. The remaining 13 mutants that expressed soluble protein were used for accurate quantification of enzymatic activity and sequencing. The mutants W15R, C19Y, R22H/K83E, P61S, S73P, S109P, and Q112R were found to have activity lower than 1% of the wild-type and were considered as “inactive mutants”, whereas the mutants K83E, Q102R, and L147F still have a large fraction of the activity and were thus considered as “partially inactivated mutants”. Molecular modeling experiments disclosed that mutations resulting in inactivation of the enzyme were found in or near the binding pocket, whereas mutations resulting in partial inactivation were distant from both substrates. The role of the residue Ser73 in the enzyme was verified by site-directed mutagenesis. The result suggested that screening inactive point mutants from a random mutagenesis library is an efficient way of identifying functional residues in enzymes.     

Characterization of KCNQ1 atrial fibrillation mutations reveals distinct dependence on KCNE1

The I(Ks) potassium channel, critical to control of heart electrical activity, requires assembly of α (KCNQ1) and β (KCNE1) subunits. Inherited mutations in either I(Ks) channel subunit are associated with cardiac arrhythmia syndromes. Two mutations (S140G and V141M) that cause familial atrial fibrillation (AF) are located on adjacent residues in the first membrane-spanning domain of KCNQ1, S1. These mutations impair the deactivation process, causing channels to appear constitutively open. Previous studies suggest that both mutant phenotypes require the presence of KCNE1. Here we found that despite the proximity of these two mutations in the primary protein structure, they display different functional dependence in the presence of KCNE1. In the absence of KCNE1, the S140G mutation, but not V141M, confers a pronounced slowing of channel deactivation and a hyperpolarizing shift in voltage-dependent activation. When coexpressed with KCNE1, both mutants deactivate significantly slower than wild-type KCNQ1/KCNE1 channels. The differential dependence on KCNE1 can be correlated with the physical proximity between these positions and KCNE1 as shown by disulfide cross-linking studies: V141C forms disulfide bonds with cysteine-substituted KCNE1 residues, whereas S140C does not. These results further our understanding of the structural relationship between KCNE1 and KCNQ1 subunits in the I(Ks) channel, and provide mechanisms for understanding the effects on channel deactivation underlying these two atrial fibrillation mutations.     

Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering

The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen. However, large-scale production of L49-sFv-bL from refolded E. coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein. Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases. One further unusual residue, found in <3% of variable sequences, was observed at position H39. Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E. coli. The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein. In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein. The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations. Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively. These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.     

Identification of key residues in subgroup A avian leukosis virus envelope determining receptor binding affinity and infectivity of cells expressing chicken or quail Tva receptor

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells ( approximately 200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.     

The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-inactivating proteins (RIPS), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y1 14F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A₁ form of Slt-IA (wild-type-). The same general relationships held: relative to wild type-, Y114F- was attenuated about 7-fold, and Y114S- about 300-fold. Tryptic digestion profiles of the mutant proteins were similar to those of the corresponding wild-type, indicating that the amino acid substitutions had not caused major alterations in conformation. We conclude that Y114 plays a significant role in the activity of Slt-IA, one which is quantitatively similar to that of Y77, and one which is predicated on the presence of both its weakly acidic phenolic hydroxyl and its aromatic ring.     

Site-directed mutagenesis and functional analysis of active site acidic amino acid residues D142, D144 and E146 in Manduca sexta (tobacco hornworm) chitinase

Chitinases (EC 3.2.1.14) are glycosyl hydrolases that catalyze the hydrolysis of beta-(1, 4)-glycosidic bonds in chitin, the major structural polysaccharide present in the cuticle and gut peritrophic matrix of insects. Two conserved regions have been identified from amino acid sequence comparisons of family 18 glycosyl hydrolases, which includes Manduca sexta (tobacco hornworm) chitinase as a member. The second of these regions in M. sexta chitinase contains three very highly conserved acidic amino acid residues, D142, D144 and E146, that are probably active site residues. In this study the functional roles of these three residues were investigated using site-directed mutagenesis for their substitutions to other amino acids. Six mutant proteins, D142E, D142N, D144E, D144N, E146D and E146Q, as well as the wild-type enzyme, were produced using a baculovirus-insect cell line expression system. The proteins were purified by anion-exchange chromatography, after which their physical, kinetic and substrate binding properties were determined. Circular dichroism spectra of the mutant proteins were similar to that of the wild-type protein, indicating that the presence of mutations did not change the overall secondary structures. E146 was required for enzymatic activity because mutants E146Q and E146D were devoid of activity. D144E retained most of the enzymatic activity, but D144N lost nearly 90%. There was a shift in the pH optimum from alkaline pH to acidic pH for mutants D142N and D144E with minimal losses of activity relative to the wild-type enzyme. The pH-activity profile for the D142E mutation resembled that of the wild-type enzyme except activity in the neutral and acidic range was lower. All of the mutant proteins bound to chitin. Therefore, none of these acidic residues was essential for substrate binding. The results indicate that E146 probably functions as an acid/base catalyst in the hydrolytic mechanism, as do homologous residues in other glycosyl hydrolases. D144 apparently functions as an electrostatic stabilizer of the positively charged transition state, whereas D142 probably influences the pKa values of D144 and E146.     

Treatment of hepatocellular carcinoma in a mouse xenograft model with an immunotoxin which is engineered to eliminate vascular leak syndrome

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin and interleukin-2 therapy. It has been evidenced that VLS-inducing molecules share a three-amino acid consensus motif, (x)D(y), which may be responsible for initiating VLS. Here we have constructed a recombinant immunotoxin (SMFv-PE38KDEL) by genetically fusing PE38KDEL to a single-chain antibody derived from SM5-1 monoclonal antibody, which has a high specificity for melanoma, hepatocellular carcinoma and breast cancer. In order to eliminate VLS induced by this PE38KDEL-based immunotoxin, a panel of mutants were generated by changing amino acid residues adjacent to its three (x)D(y) motifs in the three-dimensional structure. One of the SMFv-PE38KDEL mutants, denoted as mut1, displayed a similar protein synthesis inhibitory in a reticulocyte lysate translation assay compared to the wild-type SMFv-PE38KDEL (wt). The in vitro cytotoxicity assay indicated that mut1 specifically killed SM5-1 binding protein-positive tumor cells, although its cytotoxicity was slightly less than wt. In contrast, mut1 was shown to be much weaker in inducing VLS in mice than wt. The LD₅₀ values of wt and mut1 in mice were investigated with the result that the LD₅₀ of mut1 was about tenfold higher than that of wt. The in vivo antitumor activity of wt and mut1 were also compared in tumor-bearing nude mice. Both wt and mut1 were effective in inhibiting the tumor growth but mut1 showed improved therapeutic efficacy. These studies suggest mut1 may be a novel PE-based immunotoxin with much less toxicity for clinical use. Hao Wang, Shuichuan Song and Geng Kou contributed equally to this paper.