Evidence for a polypeptide segment at the carboxyl terminus of recombinant human gamma interferon involved in expression of biological activity

A panel of 18 murine monoclonal antibodies was raised in BALB/c mice to the full-length, 146 amino acid residue recombinant human gamma interferon (rHuIFN gamma-A). Two monoclonal antibodies, designated 47N3-6 and 30N47-1, were purified from ascites tumors and further characterized. Antibody 47N3-6 neutralized both the antiviral and antiproliferative activities of rHuIFN gamma-A. Both Western blotting and enzyme-linked immunosorbent assays indicated that antibody 47N3-6 could bind to rHuIFN gamma-A as well as to a genetically engineered truncated form lacking the first three amino-terminal residues (rHuIFN gamma-D) but did not recognize a genetically engineered variant terminating at residue 131 (rHuIFN gamma-B). This antibody also demonstrated binding to a 15 amino acid residue oligopeptide, designated F-1, corresponding to residues 132-146 at the carboxyl terminus of rHuIFN gamma-A. Chemical cleavage of peptide F-1 with cyanogen bromide produced two fragments that were separated by reversed-phase high-pressure liquid chromatography. Dot-blot analysis indicated that antibody 47N3-6 could bind to a fragment, KRKRSQHse, derived from residues 132-137 of rHuIFN gamma-A, but could bind only weakly to the cyanogen bromide fragment corresponding to residues 138-146. It was consistent with these results that antibody 47N3-6 demonstrated binding to a form lacking the five carboxyl-terminal amino acids (rHuIFN gamma-D') but did not bind to a synthetic polypeptide corresponding to residues 138-146. Peptide F-1 exhibited neither antiviral nor antiproliferative activity, and it did not antagonize the antiviral activity of rHuIFN gamma-A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

Identification of three adjacent amino acids of interleukin-2 receptor beta chain which control the affinity and the specificity of the interaction with interleukin-2.

The beta chain of the interleukin-2 (IL-2) receptor (IL-2R beta) and the interleukin-3 (IL-3) binding protein AIC2A are members of the family of cytokine receptors, which also includes the receptors for growth hormone (GHR) and prolactin. A four amino acid sequence of AIC2A has recently been shown to be critical for IL-3 binding. We analyze here the function of the analogous sequence of human IL-2R beta and identify three amino acids, Ser132, His133 and Tyr134, which play a critical role in IL-2 binding. We show that some mutant IL-2 proteins with substitutions of a critical Asp residue in the N-terminal alpha-helix bind the mutant IL-2R beta receptor with a higher affinity than the wild-type receptor. This suggests that the critical Asp34 in the ligand and the sequence Ser-His-Tyr (positions 132-134) in the receptor interact directly. On the double barrel beta-stranded structural model of cytokine receptors, the residues important for ligand binding in IL-2R beta, AIC2A and GHR map to strikingly similar locations within a barrel, with the interesting difference that it is the N-terminal barrel for GHR and the C-terminal barrel for IL-2R beta and AIC2A.     

Amino acid 310 determines the donor substrate specificity of serogroup W-135 and Y capsule polymerases of Neisseria meningitidis

The capsular polysaccharides of serogroup W-135 and Y meningococci are sialic acid-containing heteropolymers, with either galactose or glucose as the second sugar residue. As shown previously, sequences of the predicted enzymes that catalyse capsule polymerization, i.e. SiaD_W-135 and SiaD_Y, differ in only a few amino acids. By in vitro assays with purified recombinant proteins, SiaD_W-135 and SiaD_Y were now confirmed to be the capsule polymerases harbouring both hexosyltransferase and sialyltransferase activity. In order to identify amino acids crucial for substrate specificity of the capsule polymerases, polymorphic sites were narrowed down by DNA sequence comparisons and subsequent site-directed mutagenesis. Serogroup-specific amino acids were restricted to the N-terminal part of the proteins. Exclusively amino acid 310, located within the nucleotide recognition domain of the enzymes' predicted hexosyltransferase moiety, accounted for substrate specificity as shown by immunochemistry and in vitro activity assay. Pro-310 determined galactosyltransferase activity that resulted in a serogroup W-135 capsule and Gly-310 determined glucosyltransferase activity that resulted in a serogroup Y capsule. In silico analysis revealed a similar amino acid-based association in other members of the same glycosyltransferase family irrespective of the bacterial species.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

Single amino acid changes that render human IFN-alpha 2 biologically active on mouse cells.

Human IFN-alpha 1 and IFN-alpha 2 differ in 28 of 166 amino acids and show very different specific antiviral activities on human and murine cells. We have identified, by hybrid scanning and site-directed mutagenesis, three residues in IFN-alpha 2, in positions 121, 125 and 132 which, when replaced individually or jointly by their IFN-alpha 1 counterparts, modify its activity on mouse cells by up to 400-fold. We argue that these residues are involved in direct contacts with the mouse interferon receptor.     

The membrane domain of the human erythrocyte anion transport protein. Epitope mapping of a monoclonal antibody defines the location of a cytoplasmic loop near the C-terminus of the protein.

We have used synthetic peptides to study the location of the amino acid sequences in the human erythrocyte anion transport protein (band 3) which are recognized by four murine monoclonal antibodies, BRIC 130, 132, 154 and 155. These antibodies are known to react with epitopes in the protein which are on the cytoplasmic side of the membrane. The results suggest that the amino acid residues important for the reaction of BRIC 130 and BRIC 154/155 are located within amino acids 899-908 and 895-901 respectively in the cytoplasmic tail of the protein. The BRIC 132 epitope is located within amino acid residues 813-824. This is part of a surface loop in the protein which probably extends from residue 814 to residue 832 and is located on the cytoplasmic side of the membrane. These results provide direct evidence for the topographical location of a sequence in a poorly understood region of the protein.     

Mutational analysis of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) nucleotide pyrophosphatase/phosphodiesterase shows the role of six amino acids in the catalytic mechanism

Nucleotide pyrophosphatases/phosphodiesterases (NPPs, PF01663) release nucleoside 5′-monophosphates from a wide range of nucleotide substrates. Only very recently, the first plant members of the NPP family were characterised (Joye et al. J Cereal Sci 51: 326–336, 2010), and little is known about their substrate-specifying residues. We elucidated the role of six amino acid residues of the recently identified and characterised Triticum aestivum L. NPP (Joye et al. J Cereal Sci 51: 326–336, 2010). Substitution of the highly conserved catalytic Thr132 into Ser or Ala completely abolished enzyme activity. Mutation of a highly conserved His255 residue into an apolar Ala suprisingly increased enzyme activity against most phosphodiester substrates. Four other residues moderately to highly conserved over NPPs of different organisms were studied as well. Mutation of the Asn153, Asn165 and Glu199 into an Arg, Ser and Asp residue, respectively, increased the relative enzyme activity against p-nitrophenyl phosphate. Furthermore, mutation of Phe194 into Ser increased the relative enzyme activity against adenosine 5′-monophosphate-containing substrates, although the overall enzyme activity of this mutant enzyme decreased. We conclude that the structural requirements and the conservation of the amino acids of the catalytic site of TaNPPr and, by extension, probably of all NPPs, are very stringent.     

Peptide analogs from E-cadherin with different calcium-binding affinities.

Cadherins are a family of calcium-dependent cell-surface proteins that are fundamental in controlling the development and maintenance of tissues. Motif B of E-cadherin seems to be a crucial calcium-binding site as single point mutations (D134A and D134K) completely inactivate its adhesion activity. We analyzed peptide models corresponding to motif B (amino acids 128-144) as well as selected mutations of this motif. Our NMR studies showed that this motif B sequence is actually an active calcium-binding region, even in the absence of the rest of the cadherin molecule. We found that the binding affinity of this motif is very sensitive to mutations. For example, our peptide P128-144 with the native calcium-binding sequence has an affinity of Kd 0.4 mM, whereas the mutants P128-144/ D134A and P128-144/D134K containing the replacement of Asp134 by Ala and Lys, have Kd values of only 1.5 and 11 mM, respectively. Removing Asp at position 134, which correlates with the loss of adhesion activity, decreases calcium-binding affinity 20-fold. Ala132, along with residues Asp134, Asp136 and Asn143, is involved in calcium binding in solution. We also demonstrated that the calcium-binding affinity can be increased 3-fold when an additional Asp is introduced at position 132. In 50% organic solvent, this binding affinity of peptide P128-144/A132D (17-mer) from E-cadherin is similar to that of peptide P72-100/C73-77-91A (29-mer) from alpha-lactalbumin.     

Octapeptide repeat region and N-terminal half of hydrophobic region of prion protein (PrP) mediate PrP-dependent activation of superoxide dismutase

Cellular prion protein PrP(C) contains two evolutionarily conserved domains among mammals; viz., the octapeptide repeat region (OR; amino acid residue 51-90) and the hydrophobic region (HR; amino acid residue 112-145). Accumulating evidence indicates that PrP(C) acts as an inhibitor of apoptosis and regulator of superoxide dismutase (SOD) activity. To further understand how PrP(C) activates SOD and prevents apoptosis, we provide evidence here that OR and N-terminal half of HR mediate PrP(C)-dependent SOD activation and anti-apoptotic function. Removal of the OR (amino acid residue 53-94) enhances apoptosis and decreases SOD activity. Deletion of the N-terminal half of HR (amino acids residue 95-132) abolishes its ability to activate SOD and to prevent apoptosis, whereas that of the C-terminal half of HR (amino acids residue 124-146) has little if any effect on the anti-apoptotic activity and SOD activation. These data are consistent with a model in which the anti-apoptotic and anti-oxidative function of PrP(C) is regulated by not only OR but also the N-terminal half of HR.     

Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase

The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein. The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein. In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala. Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time. High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus. The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants. These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase.     

The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N.     

Structural requirements for glycosyl-phosphatidylinositol-anchor attachment in the cellular receptor for urokinase plasminogen activator.

The urokinase-plasminogen-activator receptor (u-PAR) is a glycosyl-phosphatidylinositol(glycosyl-PtdIns)-anchored membrane protein. Using site-directed mutagenesis, we have studied features in the u-PAR sequence important for successful glycosyl-PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282-Gly283-Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl-PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl-PtdIns-attachment site. In the last eight COOH-terminal amino acids encoded in u-PAR cDNA, deletion of this sequence (residues 306-313) completely prevented glycosyl-PtdIns attachment. However, the remaining COOH-terminal region proved still to possess a potential glycosyl-PtdIns signal activity; it could be converted to a new functional glycosyl-PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u-PAR to a glycosyl-PtdIns-anchored protein, indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial acquisition of the glycosyl-PtdIns-anchoring ability. These findings show that charged amino acids placed in the COOH-terminus interfere negatively with glycosyl-PtdIns-anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.     

Amino acid sequence of the signal peptide of mitochondrial nicotinamide nucleotide transhydrogenase as determined from the sequence of its messenger RNA

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase was recently deduced from isolated cDNAs and reported [Yamaguchi, M., Hatefi, Y., Trach, K., and Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767]. The cDNAs lacked the N-terminal coding region, however, and the 8 N-terminal residues were determined by protein sequencing. In the present study, the nucleotide sequence of the 5' upstream region was determined by dideoxynucleotide sequencing of the transhydrogenase messenger RNA, and amino acid sequences of the N-terminal region and the signal peptide of the enzyme were deduced from the nucleotide sequence. The N-terminal sequence of the enzyme as deduced from the mRNA sequence is the same as that determined by protein sequencing, with one difference. Protein sequencing showed Ser as the N-terminal residue. The mRNA sequence indicated that Ser is the second N-terminal residue, and the first is Cys. That preparations of the enzyme are mixtures of two polypeptides, one polypeptide being one residue shorter at the N terminus than the other, has been pointed out in the above reference. The signal peptide consists of 43 residues, is rich in basic (4 Lys, 2 Arg) and hydroxylated (4 Thr, 3 Ser) amino acids, and lacks acidic residues.     

Characterization of hen egg white- and yolk-riboflavin binding proteins and amino acid sequence of egg white-riboflavin binding protein

White- and Yolk-riboflavin binding proteins were isolated from hen eggs, and characterized as to their chemical properties. White- and Yolk-RBPs had almost same amino acid compositions except for glutamic acid, but their carbohydrate compositions were different from each other. The complete amino acid sequence of White-RBP was determined by conventional methods. White-RBP comprised 219 amino acid residues, and the amino-terminus was pyroglutamic acid (pyrrolidonecarboxylic acid). Two amino acids, lysine and asparagine, were found at the fourteenth residue from the amino-terminus. Carbohydrate chains were linked to asparagine residues at positions 36 and 147. Both White- and Yolk-RBPs were phosphorylated. In White-RBP either six or seven of nine serine residues between Ser(185) and Ser(197) were phosphorylated. The amino acid sequences around phosphoserines showed that phosphorylation might occur at a serine residue in one of the following sequences; Ser-X-Glu or Ser-X-Ser(P).     

Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.     

Serine 132 is the C3 covalent attachment point on the CH1 domain of human IgG1.

The covalent binding of C3 (complement component C3) to antigen-antibody complexes (Ag.Ab; immune complexes (ICs)) is a key event in the uptake, transport, presentation, and elimination of Ag in the form of Ag.Ab.C3b (IC.C3b). Upon interaction of C3 with IgG.IC, C3b.C3b.IgG covalent complexes are formed that are detected on SDS-polyacrylamide gel electrophoresis by two bands corresponding to C3b.C3b (band A) and C3b.IgG (band B) covalent complexes. This allows one to evaluate the covalent binding of C3b to IgG antibodies. It has been described that C3b can attach to both the Fab (on the CH1 domain) and the Fc regions of IgG. Here the covalent interaction of C3b to the CH1 domain, a region previously described spanning residues 125-147, has been studied. This region of the CH1 domain is exposed to solvent and contains a cluster of six potential acceptor sites for ester bond formation with C3b (four Ser and two Thr). A set of 10 mutant Abs were generated with the putative acceptor residues substituted by Ala, and we studied their covalent interaction with C3b. Single (Ser-131, Ser-132, Ser-134, Thr-135, Ser-136, and Thr-139), double (positions 131-132), and multiple (positions 134-135-136, 131-132-134-135-136, and 131-132-134-135-136-139) mutants were produced. None of the mutants (single, double, or multiple) abolished completely the ability of IgG to bind C3b, indicating the presence of C3b binding regions other than in the CH1 domain. However, all mutant Abs, in which serine at position 132 was replaced by Ala, showed a significant decrease in the ability to form C3b.IgG covalent complexes, whereas the remaining mutants had normal activity. In addition we examined ICs using the F(ab')2 fragment of the mutant Abs, and only those containing Ala at position 132 (instead of Ser) failed to bind C3b. Thus Ser-132 is the binding site for C3b on the CH1 domain of the heavy chain, in the Fab region of human IgG.     

A single amino acid determinant of the membrane localization of lipoproteins in E. coli

When beta-lactamase was fused with the signal peptide plus the amino-terminal 9 amino acid residues of the major outer membrane lipoprotein, the resultant lipo-beta-lactamase (LL-1) was shown to be localized to the outer membrane. However, when the 9 residue sequence was replaced with the amino-terminal 12 residue sequence of lipoprotein-28, an inner membrane protein, the resultant lipo-beta-lactamase (LL-2) was found exclusively in the inner membrane. The localization of LL-2 was shifted to the outer membrane simply by substituting the second amino acid residue (Asp) of LL-2 with Ser. Conversely, the alteration of the second residue (Ser) of LL-1 to Asp resulted in the localization of LL-1 to the inner membrane. These results suggest that the second amino acid residue of the lipoproteins plays a crucial role in determining their final locations in the E. coli envelope.