Sixth Residue Research Articles

Consensus sequence for precursor processing at mono-arginyl sites. Evidence for the involvement of a Kex2-like endoprotease in precursor cleavages at both dibasic and mono-arginyl sites.

Many peptide hormones and neuropeptides are produced from larger, inactive precursors through endoproteolysis at sites usually marked by paired basic residues (primarily Lys-Arg and Arg-Arg), or occasionally by a monobasic residue (primarily Arg). Based upon data concerning processing of prorenin and its mutants around the native Lys-Arg cleavage site expressed in mouse pituitary AtT-20 cells, we present the following sequence rules that govern mono-arginyl cleavages: (a) a basic residue at the fourth (position -4) or the sixth (position -6) residue upstream of the cleavage site is required, (b) at position -4, Arg is more favorable than Lys, and (c) at position 1, a hydrophobic aliphatic residue is not suitable. These rules are compatible with those proposed by comparison of precursor sequences around mono-arginyl cleavage sites. We also provide evidence that precursor cleavages at mono-arginyl and dibasic sites can be catalyzed by the same Kex2-like processing endoprotease, PC1/PC3.

A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating

The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.

A hybrid ribonuclease H. A novel RNA cleaving enzyme with sequence-specific recognition.

A hybrid enzyme which site-specifically hydrolyzes RNA was created by covalently linking an oligodeoxyribonucleotide to Escherichia coli ribonuclease HI, an enzyme which specifically cleaves RNA moiety of DNA/RNA hybrids. A cysteine residue was substituted for Glu135 by site-directed mutagenesis in the mutant enzyme, in which all 3 free cysteine residues were replaced by alanine (Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Biochem. J. 271, 59-66), and coupled with a maleimide group, which is attached to the 5' terminus of the nonadeoxyribonucleotide (5'-GTCATCTCC-3') with a flexible tether. The resulting hybrid enzyme, d9-C135/RNase H, cleaved the phosphodiester bond between the fifth and sixth residues of the complementary nonaribonucleotide, without addition of the oligodeoxyribonucleotide. The nonaribonucleotide is cleaved by the wild-type or unmodified mutant enzyme only when the complementary oligodeoxyribonucleotide is present. When the kinetic parameters of the hybrid enzyme for the hydrolysis of the nonaribonucleotide were compared with those of the unmodified mutant enzyme for the hydrolysis of the nonanucleotide duplex, the hybrid enzyme exhibited a 7- and 4-fold decreases in the Km and kcat values, respectively, indicating that it performs multiple turnovers and has a sufficiently high hydrolytic activity. Hybrid ribonucleases H with various oligodeoxyribonucleotides in size and sequence, therefore, might be used as excellent tools for structural and functional studies of RNA.

Maternally Transmitted Antigen of Mice: A Model Transplantation Antigen

Molecular identification proved Mta, the maternally transmitted antigen of mice, to be a model minor histocompatibility (H) antigen. It consists of a peptide, MTF, that is presented on the cell surface by an H-2 class-I molecule, HMT. MTF is derived from ND1, a mitochondrially encoded protein, and the amino-terminal N-formyl-methionine is essential for binding to HMT; conservative substitutions at the sixth residue causes MTF to be a minor H antigen. HMT is encoded by the M3 gene at the telomeric end of the H-2 complex. The peptide-binding site of HMT is hydrophobic, and allelic forms of the mature protein differ by only three amino acids. Homologues and analogues of the mouse Mta system have recently been identified in rats.

Complementing mutant alleles define three loci involved in mannosylation of Man5-GlcNAc2-P-P-dolichol in Chinese hamster ovary cells

Dolichol-linked oligosaccharides consisting of two N-acetylglucosamine, nine mannose, and three glucose residues (Glc3Man9GlcNAc2) are transferred to proteins that contain the consensus sequence Asn-X-Ser/Thr. This transfer occurs upon protein import into the lumen of the endoplasmic reticulum. An intermediate in the biosynthesis of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide contains two GlcNAc and five mannose residues. This intermediate serves as a substrate for further mannosylation and glucosylation before transfer to protein. The addition of the sixth mannose residue to this intermediate requires the enzyme mannosyltransferase VI and the mannose donor, mannose-P-dolichol. Several different CHO cell line mutants that fail to efficiently catalyze this transfer have been described. In this report, we examine seven independent mutant cell lines with various biochemical phenotypes and demonstrate that all can be assigned to one of three genetic complementation groups. One mutation affects mannose-P-dolichol biosynthesis (Lec15), three affect dolichol phosphate biosynthesis (Lec9), and three appear to affect the functional orientation of enzyme substrates (PIR).

Investigation of the structural parameters involved in the .mu. and .delta. opioid receptor discrimination of linear enkephalin-related peptides

The previous rules proposed for selective recognition of mu and delta opioid receptors by modified enkephalins were investigated through an extensive structure-activity study. Thus, modifications of the sequence of TRIMU 4 (Tyr-D-Ala-Gly-NHCH(CH3)CH2CH(CH3)2, a peptide that exhibits mu selectivity close to that of DAGO (Try-D-Ala-Gly-N(Me)Phe-Gly.ol), were performed for two positions, 2 and 4, critical for mu recognition. The drastic loss of potency following introduction of L-Ala or Aib in position 2 emphasizes the importance of the stereochemistry and the steric size of the X2 amino acid for optimal mu binding. The enhancement of the intrinsic flexibility of the C-terminal alkyl chain of TRIMU 4 through removal of a methyl group leads to TRIMU 5 (Tyr-D-Ala-Gly-NHCH2CH2CH(CH3)2), a peptide with a mu selectivity similar to that of DAGO. In contrast, introduction of an O-tert-butyl Ser2 residue increases affinity for delta receptors. In the hexapeptide series derived from DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr), a D-Thr2 moiety was shown to be very efficient in improving delta recognition and delta selectivity appeared also to be modulated by the nature of the sixth residue. The potencies of the 24 peptides studied to inhibit the electrically evoked contractions of the GPI or MVD are relatively well correlated with their affinities for brain mu or delta receptors labeled with [3H]DAGO or [3H]DSLET, respectively. Moreover, the analgesic potency (hot plate test) of the peptides is related to their affinity for rat brain mu receptors. The wide range of receptor affinities exhibited by the compounds reported here could be useful to study the physiological role of mu and delta receptors.

Structural features of a rye-bran arabinoxylan with a low degree of branching

Two fractions (AX-1 and AX-II) of an l-arabino- d-xylan with a low degree of branching were isolated from rye bran by different extraction procedures with and without a chlorite-delignification step. The yields and compositions of AX-I and AX-II were similar, but the d.p. of AX-II was ∼100 units higher. AX-I was shown by partial hydrolysis, methylation analysis, and 13C-n.m.r. spectroscopy to contain a backbone of (1→4)-linked β- d-xylopyranosyl residues substituted with single α- l-arabinofuranosyl groups at position 3 of every sixth or seventh d-xylosyl residue.

Contents of volume 9

Crude methanol extracts of fresh skins of the European discoglossid frogs Alytes obstetricans, Bombina bombina, and Bombina variegata were subjected to chromatography on alumina columns and to gel filtration on Sephadex columns. The active constituents of these extracts, the peptides alytesin and bombesin, were obtained in pure form. Acid hydrolysis, digestion with trypsin and chymotrypsin, and group determination experiments demonstrated that alytesin and bombesin are tetradecapeptides differing only in the second and sixth residues from the NH2-terminus.Synthesis, carried out only for bombesin, has confirmed the proposed sequence.The similarity between the structure of alytesin and bombesin on the one hand and that of ranatensin from Rana pipiens on the other is noted and discussed.

Structures of enzyme-substrate complexes of lysozyme

Conformational energy calculations were used to determine the binding structures of two oligosaccharides (GlcNAc)(n), in which n = 5 and 6, in the rigid active site of lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1.17). Starting with the lowest energy binding structures of (GlcNAc)(4) as determined in a previous publication, we added a fifth GlcNAc residue to this tetramer in three different conformations, corresponding to the left-handed and right-handed helical structures and an intermediate structure, and the energy of each complex was minimized. The most stable binding conformation of the fifth residue of the pentamer was closest to the left-handed helical one. During energy minimization, the fourth residue of the pentamer moved from its initial position near the surface of the active site farther into the active site cleft at binding site D. Binding structures of (GlcNAc)(6) were then examined by addition of a residue to the lowest energy structure of (GlcNAc)(5), and it was found that the sixth residue of the hexamer binds in a conformation again close to the left-handed helical one. Stable binding regions of the rigid active site for the fifth and sixth residues were found to be near arginyl 45 and asparaginyl 46, on the opposite side of the active site cleft from arginyl 114. When the calculated structure of the lysozyme-(GlcNAc)(4) complex (used here as the starting structure for addition of the fifth and sixth residues) is compared with recent experimental data, it is found that the calculated structure is a reasonable one. Of all binding regions available to the saccharide residues, the C site binds GlcNAc with the lowest energy, in agreement with experiments.

The influence of nearest-neighbor amino acids on the conformation of the middle amino acid in proteins: comparison of predicted and experimental determination of -sheets in concanavalin A.

A 20 x 20 table of tripeptides has been compiled that may be used to locate beta-sheet breaking and alpha-helix breaking residues in proteins. It is based on the definition of an alpha-helical and a beta-sheet domain on the (varphi, Psi) map based on the occurrences of alpha-helices and beta-sheets in 12 known proteins whose sequence and three-dimensional structure have been determined. Each entry in the 20 x 20 table lists three numbers, the frequency of occurrences of the middle amino acid (n) in relation to its nearest neighbors (n - 1) and (n + 1) in the alpha-helical domain, the beta-sheet domain and outside these regions. The regions between two beta-sheet-breaking residues would be permissively beta-sheet regions. The sequence of concanavalin A has been examined in this manner and of the 13 beta-strands defined by x-ray crystallography, 10 were in agreement with the permissively beta-sheet regions and, in the remaining three, beta-sheet-breaking residues were the third in one, and the third, fourth, and fifth residues in another, and the sixth residue in the third from the beginning of the beta-strands. The findings provide strong support for the role of nearest-neighboring amino acids in determining secondary structure of proteins.

The primary structure of a monoclonal human lambda-type immunoglobulin L-chain of subgroup II (Bence-Jones protein NEI).

Protein NEI, which has a molecular weight of 23 500 and contains 216 amino acids, was isolated from the urine of a patient with multiple myeloma by salt-precipitation and purified by ion-exchange chromatography, block electrophoresis and gel filtration. 23 tryptic peptides have been isolated from the aminoethylated protein and 13 chymotryptic peptides from the carboxymethylated protein by ion-exchange chromatography. Sequence studies have mainly been carried out with the tryptic peptides; the chymotryptic peptides have been used for overlaps in the variable region, since the alignment of the tryptic peptides by homology was not unambiguous. Protein NEI belongs to subgroup II of the λ-chains. It has a series of subgroup specific exchanges and a subgroup specific deletion at position 96. Protein NEI differs from all other λ-chains of subgroup II by possessing a sixth cysteine residue. This additional cysteine residue at position 88 is so far unique, as it is adjacent to an invariant cysteine at position 87. Protein NEI also contains a carbohydrate moiety attached to an aspartic acid or asparagine (Asx) residue at position 93, one of the hypermutable areas of the variable part. Furthermore we conclude by comparison with other λ-chains that all completely elucidated λ-chains can be ordered into four subgroups. The subgroups and linkage groups within subgroups are in complete agreement with an evolutionary origin of antibody variability and are incompatible with somatic hypermutation models.

Isolation and amino acid sequences of alytesin and bombesin, two analogous active tetradecapeptides from the skin of European discoglossid frogs

Crude methanol extracts of fresh skins of the European discoglossid frogs Alytes obstetricans, Bombina bombina, and Bombina variegata were subjected to chromatography on alumina columns and to gel filtration on Sephadex columns. The active constituents of these extracts, the peptides alytesin and bombesin, were obtained in pure form. Acid hydrolysis, digestion with trypsin and chymotrypsin, and group determination experiments demonstrated that alytesin and bombesin are tetradecapeptides differing only in the second and sixth residues from the NH 2-terminus. Synthesis, carried out only for bombesin, has confirmed the proposed sequence. The similarity between the structure of alytesin and bombesin on the one hand and that of ranatensin from Rana pipiens on the other is noted and discussed.

α-1, 4-Glucan: α-1, 4-Glucan 6-glycosyl transferase from trout liver

1. 1. α-1, 4-Glucan: α-1, 4-glucan 6-glycosyl transferase was purified about 200-fold from trout liver. 2. 2. The activity of the enzyme is distinguished from that of α- and β-amylases and it is shown to increase the β-limit of glycogen wherein the exterior chains have been elongated. 3. 3. The enzyme is activated by various cations, but unaffected by fluoride, phosphate, and the smaller malto-oligosaccharides. 4. 4. In addition to amylopectin the enzyme will also ramify ainylose and the β-limit dextrin of amylopectin. 5. 5. Determination of the structure of native trout liver glycogen suggests that the action of the enzyme is to remove an oligosaccharide not more than 8–9 glucose residues long from the non-reducing end of an elongated chain and attach it by an α-1,6-linkage onto the fifth or sixth glucose residue of an adjacent chain.

Haemoglobin F Texas II (alpha-2 gamma-2, 6 Glu-Lys), the second of the haemogloin F Texas variants.

Summary A survey of 13,000 cord bloods in Great Britain yielded an electrophoretically abnormal variant of Haemoglobin F which resembled Haemoglobin F Texas. Haemoglobin F Texas might be either α2γ25G1u→Lys or α2γ26Glu→Lys. The first has been described and was named Haemoglobin F Texas I. The present variant has the same substitution in the sixth residue of the γ chain and is designated as Haenioglobin F Texas II.

HEMOGLOBIN F TEXAS: GAMMA-CHAIN VARIANT.

An abnormal fetal hemoglobin, designated hemoglobin F(Texas), was found in the cord blood of five Negro infants (all related and three of them siblings) and of one Caucasian infant. Studies on the chemical structure of this variant indicate that there is a substitution of a lysyl residue for either the fifth or sixth glutamyl residue of the gamma-chain.