Large hepatitis delta antigen in packaging and replication inhibition: role of the carboxyl-terminal 19 amino acids and amino-terminal sequences.

Hepatitis delta virus (HDV) encodes two proteins, the small delta antigen (SHDAg) and large delta antigen (LHDAg). The latter is identical to the former except for the presence of additional 19 amino acids at the C terminus. While SHDAg is required for HDV replication, LHDAg inhibits replication and, together with hepatitis B surface antigen (HBsAg), is required for the assembly of HDV. The last 19 C-terminal amino acids of LHDAg are essential for HDV assembly. Most of LHDAg (amino acids 19 to 146 and 163 to 195) had been shown to be dispensable for packaging with HBsAg. To discern whether the last 19 C-terminal amino acids solely constitute the signal for packaging with HBsAg, we constructed two LHDAg deletion mutants and tested their abilities to be packaged with HBsAg in cotransfection experiments. We found that deletion of amino acids 2 to 21 and 142 to 165 did not affect LHDAg packaging. This result suggested that only the last 19 C-terminal amino acids of LHDAg are required for packaging. We further constructed two plasmids which expressed c-H-ras with or without additional 19 C-terminal amino acids identical to those in LHDAg. Only c-H-ras with additional 19 amino acids could be cosecreted with HBsAg in the cotransfection experiment. This result confirmed that the C-terminal 19 amino acids are the packaging signal for HBsAg. We also tested the trans activation activity and trans-dominant inhibitory activity of the deletion mutants of SHDAg and LHDAg, respectively. In contrast to deletion of amino acids 142 to 165, deletion of amino acids 2 to 21 impaired the trans-dominant inhibitory activity of LHDAg. Deletion of amino acids 2 to 21 and 142 to 165 did not affect the trans activation activity of SHDAg. This result suggested that a functional domain which is important for the trans-dominant inhibitory activity of LHDAg exists in the amino terminus of HDAg.     

Adaptation of the tRNA population of maize endosperm for zein synthesis.

Maize endosperm, 30 days after pollination is actively synthesizing zein, a storage protein containing high amounts of glutamine. leucine and alanine. Endosperm tRNAs have a higher accepting activity than embryo tRNAs for these three amino acids, but not for some other (control) amino acids. This increase in accepting activity is accompanied by a change in the distribution of the isoaccepting tRNA species corresponding to these three amino acids, but not of the isoacceptors corresponding to some other (control) amino acids. These results are in favor of the theory of functional adaptation of tRNA population.     

Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD13): influence of N-linked glycosylation

Aminopeptidase N (APN), a 150-kDa metalloprotease also called CD13, serves as a receptor for serologically related coronaviruses of humans (human coronavirus 229E [HCoV-229E]), pigs, and cats. These virus-receptor interactions can be highly species specific; for example, the human coronavirus can use human APN (hAPN) but not porcine APN (pAPN) as its cellular receptor, and porcine coronaviruses can use pAPN but not hAPN. Substitution of pAPN amino acids 283 to 290 into hAPN for the corresponding amino acids 288 to 295 introduced an N-glycosylation sequon at amino acids 291 to 293 that blocked HCoV-229E receptor activity of hAPN. Substitution of two amino acids that inserted an N-glycosylation site at amino acid 291 also resulted in a mutant hAPN that lacked receptor activity because it failed to bind HCoV-229E. Single amino acid revertants that removed this sequon at amino acids 291 to 293 but had one or five pAPN amino acid substitution(s) in this region all regained HCoV-229E binding and receptor activities. To determine if other N-linked glycosylation differences between hAPN, feline APN (fAPN), and pAPN account for receptor specificity of pig and cat coronaviruses, a mutant hAPN protein that, like fAPN and pAPN, lacked a glycosylation sequon at 818 to 820 was studied. This sequon is within the region that determines receptor activity for porcine and feline coronaviruses. Mutant hAPN lacking the sequon at amino acids 818 to 820 maintained HCoV-229E receptor activity but did not gain receptor activity for porcine or feline coronaviruses. Thus, certain differences in glycosylation between coronavirus receptors from different species are critical determinants in the species specificity of infection.     

Truncation of NH2-terminal amino acid residues increases agonistic potency of leukotactin-1 on CC chemokine receptors 1 and 3

Leukotactin-1 (Lkn-1) is a human CC chemokine that binds to both CC chemokine receptor 1 (CCR1) and CCR3. Structurally, Lkn-1 is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2) terminus, and contains two extra cysteines. NH(2)-terminal amino acids of Lkn-1 were deleted serially, and the effects of each deletion were investigated. In CCR1-expressing cells, serial deletion up to 20 amino acids (Delta20) did not change the calcium flux-inducing activity significantly. Deletion of 24 amino acids (Delta24), however, increased the agonistic potency approximately 100-fold. Deletion of 27 or 28 amino acids also increased the agonistic potency to the same level shown by Delta24. Deletion of 29 amino acids, however, abolished the agonistic activity almost completely showing that at least 3 amino acid residues preceding the first cysteine at the NH(2) terminus are essential for the biological activity of Lkn-1. Loss of agonistic activity was due to impaired binding to CCR1. In CCR3-expressing cells, Delta24 was the only form of Lkn-1 mutants that revealed increased agonistic potency. Our results indicate that posttranslational modification is a potential mechanism for the regulation of biological activity of Lkn-1.     

Chemotopy of amino acids on the olfactory bulb predicts olfactory discrimination capabilities of zebrafish Danio rerio

Amino acids reliably evoke strong responses in fish olfactory system. The molecular olfactory receptors (ORs) are located in the membrane of cilia and microvilli of the olfactory receptor neurons (ORNs). Axons of ORNs converge on specific olfactory bulb (OB) glomeruli and the neural responses of ORNs expressing single Ors activate glomerular activity patterns typical for each amino acid. Chemically similar amino acids activate more similar glomerular activity patterns then chemically different amino acids. Differential glomerular activity patterns are the structural basis for amino acid perception and discrimination. We studied olfactory discrimination in zebrafish Danio rerio (Hamilton 1822) by conditioning them to respond to each of the following amino acids: L-Ala, L-Val, L-Leu, L-Arg, and L-Phe. Subsequently, zebrafish were tested for food searching activities with 18 nonconditioned amino acids. The food searching activity during 90 s of the test period was significantly greater after stimulation with the conditioned stimulus than with the nonconditioned amino acid. Zebrafish were able to discriminate all the tested amino acids except L-Ile from L-Val and L-Phe from L-Tyr. We conclude that zebrafish have difficulties discriminating amino acid odorants that evoke highly similar chemotopic patterns of activity in the OB.     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

Measurement and subcellular distribution of choloyl-CoA synthetase and bile acid-CoA:amino acid N-acyltransferase activities in rat liver

An improved method for assaying choloyl-CoA synthetase activity (E.C. 6.2.1.7) and two methods for specific measurement of bile acid-CoA:amino acid N-acyltransferase activity (E.C. 2.3.1) are described. The methods are shown to be reproducible, linear with respect to time and enzyme protein, and result in estimates of enzymic activity that conform to the theoretical stoichiometry of the individual reactions. Utilizing these methods, the subcellular distribution of the rat liver enzymic activity catalyzing the formation of glycine and taurine conjugates of bile acids is shown. Choloyl-CoA synthetase is associated with the microsomal membranes and bile acid-CoA:amino acid N-acyltransferase activity with the postmicrosomal supernatant. No significant amino acid N-acyltransferase activity is present in the lysosome fraction. These studies provide methods that will permit further study of the individual enzymic reactions involved in the intrahepatic conjugation of bile acids with amino acids.     

Activity-dependent reversible inactivation of the general amino acid permease

The general amino acid permease, Gap1p, of Saccharomyces cerevisiae transports all naturally occurring amino acids into yeast cells for use as a nitrogen source. Previous studies have shown that a nonubiquitinateable form of the permease, Gap1p(K9R,K16R), is constitutively localized to the plasma membrane. Here, we report that amino acid transport activity of Gap1p(K9R,K16R) can be rapidly and reversibly inactivated at the plasma membrane by the presence of amino acid mixtures. Surprisingly, we also find that addition of most single amino acids is lethal to Gap1p(K9R,K16R)-expressing cells, whereas mixtures of amino acids are less toxic. This toxicity appears to be the consequence of uptake of unusually large quantities of a single amino acid. Exploiting this toxicity, we isolated gap1 alleles deficient in transport of a subset of amino acids. Using these mutations, we show that Gap1p inactivation at the plasma membrane does not depend on the presence of either extracellular or intracellular amino acids, but does require active amino acid transport by Gap1p. Together, our findings uncover a new mechanism for inhibition of permease activity in response to elevated amino acid levels and provide a physiological explanation for the stringent regulation of Gap1p activity in response to amino acids.     

Structure-function analysis of human protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1, POMGnT1

Protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) catalyzes the transfer of GlcNAc to O-mannose of glycoproteins. Mutations in the POMGnT1 gene cause a type of congenital muscular dystrophy called muscle-eye-brain disease (MEB). We evaluated several truncated mutants of POMGnT1 to determine the minimal catalytic domain. Deletions of 298 amino acids in the N-terminus and 9 amino acids in the C-terminus did not affect POMGnT1 activity, while larger deletions on either end abolished activity. These data indicate that the minimal catalytic domain is at least 353 amino acids. Single amino acid substitutions in the stem domain of POMGnT1 from MEB patients abolished the activity of the membrane-bound form but not the soluble form. This suggests that the stem domain of the soluble form of POMGnT1 is unnecessary for activity, but that some amino acids play a crucial role in the membrane-bound form.     

Interference by branched-chain amino acids in the assay of alanine with alanine dehydrogenase.

Commercial preparations of alanine dehydrogenase from Bacillus subtilis are contaminated to varying extents with activity towards branched-chain amino acids. The Km values for these amino acids are of the same order as for L-alanine (about 10(-3)M). The branched-chain amino acid dehydrogenase activity is lost on dialysis for 2--4h against water or 2mM-EDTA.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine preferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

Behavioral reactions of the clawed toad Xenopus laevis to L- and D-isomers of amino acids

Studies on the action of L- and D-isomers of amino acids upon motor activity in the clawed toad revealed that most effective among them are basic amino acids (all of them being essential) and hydrophobic amino acids (mainly essential ones). Positive correlation was found between the effectiveness of hydrophobic amino acids on the motor activity of animals and their effectiveness with respect to taste receptors. The data obtained indicate the role of the olfactory receptors in realization of the behavioural reactions of the clawed toads to amino acids.     

Modeling the solubility and activity of amino acids with the LCCI method

Summary The linear combinations of connectivity indices method (LCCI) is here employed to model the water solubility and activity of 19 natural amino acids. Starting with the molecular connectivity indices, reciprocal and supra molecular connectivity indices are designed to model the solubility and activity spaces of the natural amino acids. The reciprocal and supra molecular reciprocal connectivity indices have been obtained following the variability of the connectivity indices along solubility space of the natural amino acids. A linear combination of the reciprocals of the connectivity indices (LCRCI) showed a satisfactory modelling of the solubility and activity space while a model based on the LCRCI together with the introduction of supra reciprocal molecular connectivity indices for Pro, Ser and Arg achieved an optimal modelling of the solubility and activity space of the natural amino acids. Because the properties are a consequence of the structure (Kier and Hall, 1986)     

Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity.

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N-terminal and C-terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C-terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N-terminal and C-terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C-terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C-terminus of this sequence. Interestingly, the inactive C-terminal mutants could be complemented by cotransfection with a wild-type C-terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C-terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C-terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.     

T-Cell Antigen Receptor-alpha Chain Transmembrane Peptides: Correlation between Structure and Function

Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain that is capable of inhibiting the immune response both in vitro and in animal models of T cell mediated inflammation. CP contains two basic amino acids (lysine and arginine) in its sequence. The presence of these charged residues interspersed between hydrophobic amino acids is important for function. Here in an attempt to understand CP’s biophysical properties leading to activity we have synthesized a number of CP analogues and correlated their model structure with their biological activity. It became apparent that it is not only the charge of the amino acids but also the nature of the polar amino acids themselves and the topography and spacing between them by hydrophobic amino acids, creating a hydrophobic face, that are critical for CP function.Australian Peptide Conference Issue.     

The C-terminus of glycosylphosphatidylinositol-specific phospholipase D is essential for biological activity

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) (EC 3.1.4.50) from mammalian serum is a 115 kDa glycoprotein consisting of 816 amino acids. We found that C-terminal deletions of only two to five amino acids reduced GPI-PLD enzymatic activity by roughly 70% as compared to wild-type protein. C-terminal deletions of more than five amino acids resulted in a complete loss of GPI-PLD enzymatic activity. Point mutations at position 811 indicate that Tyr-811 may play a major role in maintaining the biological activity of GPI-PLD.     

Localization of C-terminal domains required for the maximal activity or for determination of substrate preference of maize branching enzymes

Previous analysis of a chimeric enzyme mBEII-IBspHI, in which the C-terminal 229 amino acids of maize endosperm branching enzyme isoform II (mBEII) are replaced by the corresponding 284 amino acids of isoform I (mBEI), suggested that the carboxyl terminus of maize branching enzymes may be involved in catalytic efficiency and substrate preference. In the present study, additional hybrids of mBEI and mBEII were generated and expressed in Escherichia coli BL21 (DE3) to dissect the structure/function relationships of the C-terminal regions of maize branching enzymes. A truncated form of purified mBEII-IBspHI, which lacks the C-terminal 58 amino acids, retained similar levels of V(max) in branching activity, K(m) for reduced amylose AS 320, and substrate preference for amylose than amylopectin when compared to mBEII-IBspHI. This indicates that the C-terminal extension derived from mBEI is not required for either catalysis or substrate preference. However, deletion of an additional 87 amino acids from the carboxyl terminus resulted in complete loss of activity. Replacement of the deleted C-terminal additional 87 amino acids with the corresponding 79 amino acids from mBEII restored 25% of the mBEII-IBspHI branching activity without altering substrate preference. It thus appears that a C-terminal region encompassing Leu649-Asp735 of mBEII-IBspHI is required for maximum catalytic efficiency. Another C-terminal region, residues Gln510-Asp648, of mBEII-IBspHI (Gln476-Asp614 of mBEI) may be involved in substrate-preference determination.     

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.