The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.     

The legumin gene family: structure of a B type gene of Vicia faba and a possible legumin gene specific regulatory element.

The field bean, Vicia faba L. var. minor, possesses two sub-families of 11 S legumin genes named A and B. We isolated from a genomic library a B-type gene (LeB4) and determined its primary DNA sequence. Gene LeB4 codes for a 484 amino acid residue prepropolypeptide, encompassing a signal peptide of 22 amino acid residues, an acidic, very hydrophilic alpha-chain of 281 residues and a basic, somewhat hydrophobic beta-chain of 181 residues. The latter two coding regions are immediately contiguous, but each is interrupted by a short intron. Type A legumin genes from soybean and pea are known to have introns in the same two positions, in addition to an extra intron (within the alpha-coding sequence). Sequence comparisons of legumin genes from these three plants revealed a highly conserved sequence element of at least 28 bp, centered at approximately 100 bp upstream of each cap site. The element is absent from the equivalent position of all non-legumin and other plant and fungal genes examined. We tentatively name this element "legumin box" and suggest that it may have a function in the regulation of legumin gene expression.     

The conserved methionine residue of the metzincins: a site-directed mutagenesis study.

The metalloprotease clan of the metzincins derive their name from the presence of a conserved methionine residue that is located on the C-terminal side of the zinc-binding consensus sequence HEXXHXXGXXH. This methionine residue is located in a rather divergent part of the primary sequence but is structurally very well conserved. It is located under the pyramidal base of the three histidine residues that coordinate the catalytic zinc ion and is not involved in any direct contact with the metal nor the substrate. In order to clarify its role, this methionine residue (M226) of the protease C from Erwinia chrysanthemi has been mutated to various other amino acids. The mutants M226L, M226A, M226I were sufficiently stable to be isolated, while the mutants M226H, M226S and M226N could not be purified. The kinetic properties of these mutants were analysed. All mutants showed decreased activity, whereby increases in K(M) as well as decreases in k(cat) were observed. The M226L mutant and M226C-E189 K double mutant, which has the catalytic glutamic acid substituted as well, could be crystallised. The structure of the M226L mutant was determined to a resolution of 2.0 A and refined to R(free) of 0.20. The structure is isomorphous to the wild-type and does not show large differences, with the exception of a very small movement of the zinc-liganding histidine residues. The M226C-E189 K double mutant crystal structure has been refined to an R(free) of 0.20 at 2.1 A resolution. A small rearrangement of the zinc-liganding histidine residues can be detected, which leads to a slightly different zinc coordination and could explain the decrease in activity.     

Heterosis and components of genetic variation for protein and lysine content in some grain sorghums

Summary Twelve varieties representing six geographical regions and nine taxonomic groups from a World Collection ofSorghum were used in a diallel and line X tester analysis of the nature of genetic variation for protein and lysine content. In a majority of crosses, heterosis was negative for protein, and positive for lysine.Analysis of combining ability indicated that both additive and non-additive variation were important for protein, while the non-additive component was predominant for lysine. Heterosis inlow Xlow andmedium Xlow crosses also indicated the presence of substantial non-allelic gene interactions for both the characters.Associations between protein, lysine and yield in parents were significantly different from those in the hybrids indicating considerable scope for genetic manipulation. Negative correlations between protein and lysine (% protein) and between protein and grain yield were very low. The results indicated that a high level of protein and moderately high lysine may be incorporated in high yielding varieties of sorghum, by using Caudatum kaura, Roxburghii shallu and durra from Nigeria and Sudan in the hybridisation programme.     

Purification and subunit structure of legumin of Vicia faba L. (broad bean)

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (alpha) and had a molecular weight of 37000, and three were basic (beta) with molecular weights of 20100, 20900 and 23800, were identified. The alpha and beta subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an alpha(6)beta(6) subunit model was proposed for legumin.     

The effect of carboxymethylating a single methionine residue on the subunit interactions of glycophorin A.

Human red cell glycophorin A shows an equilibrium between dimeric and monomeric forms which have been disignated PAS-1 and PAS-2, respectively. This equilibrium, which is dependent upon protein concentration is achieved by incubation in sodium dodecyl sulfate solutions at elevated temperatures and is assayed by sodium dodecyl sulfate gel electrophoresis. Carboxymethylation of glycophorin A in guanidine hydrochloride or urea alters the interactions between polypeptide chains so that the lower molecular weight form (PAS-2) is obtained much more readily. If the carboxymethylation is performed at pH 3.0 the reaction is limited to the two methionine residues of glycophorin A which are located at positions 8 and 81 in the sequence. In the presence of sodium dodecyl sulfate, only one of the two methionine residues is carboxymethylated, and glycoprotein modified under these conditions does not exhibit the change in electrophoretic mobility. Experiments with [1-14C]iodoacetic acid demonstrated that Met-81, located in the hydrophobic domain of the protein, is the residue protected by sodium dodecyl sulfate. Modification of Met-81 destabilizes the dimeric form relative to the monomer by weakening the interactions between polypeptide chains. The experiments described in this paper confirm that the hydrophobic domain of glycophorin A is involved in subunit interactions and that Met-81 plays a critical role in those interactions.     

Conversion of lysine 91 to methionine or glutamic acid in human choriogonadotropin alpha results in the loss of cAMP inducibility.

Human choriogonadotropin (hCG) is a heterodimeric glycoprotein hormone. The alpha subunit comprises 92 amino acids, of which 6 are Lys residues (Morgan, F.G., Birken, S., and Canfield, R.E. (1975) J. Biol. Chem. 250, 5247-5258). Our photoaffinity-labeling studies indicate that several of these Lys residues make contact with the lutropin receptor and are covalently cross-linked to the receptor. Lys-91 of the alpha subunit is of interest because deletion of the two alpha C-terminal residues, Lys-91 and Ser-92, results in a significant reduction in the bioactivity of lutropin and thyrotropin (Cheng, K.-W., Glazer, A.N., and Pierce, J.G. (1973) J. Biol. Chem. 248, 7930-7937). To determine the importance of Lys-alpha 91, we substituted it with Arg, Met, or Glu. The resulting mutant alpha cDNA constructs were co-transfected into CHO cells with the wild type hCG beta cDNA construct. Secreted hCG dimers were assayed for binding to receptors on porcine granulosa cells and stimulation of cAMP synthesis in a murine Leydig tumor cell line. The natural hCG, wild type hCG, and all mutant hCGs recognized the receptor, although with somewhat divergent affinities. However, there was a striking difference in the ability of cAMP induction. The natural hCG, wild type hCG, and Lys-91----Arg mutant hCG induced cAMP synthesis, whereas the Lys-91----Met and Lys-91----Glu mutants did not. These results demonstrate that Lys-91 is important for receptor modulation in the stimulation of cAMP synthesis.     

The binding of calcium to fibrinogen: some structural features.

Experiments are described which suggest that structural features are related to the existence of three high affinity calcium-binding sites in the fibrinogen molecule. The circular dichroism spectra analysis shows that the binding of calcium to this protein does not entail an overall conformational change. However several calcium-induced protective effects may be observed: 1. At pH 5.0 calcium-free fibrinogen is slightly acid-denatured. This denaturation is counteracted by the presence of calcium, whereas magnesium ions have no effect. 2. A temperature transition shift of 3 degrees C is measured in the presence of bound calcium during thermal denaturation, whereas magnesium ions have no effect. 3. Resistance to proteolysis by plasmin is observed when calcium is bound to fibrinogen. The velocity of the splitting of the earliest plasmin-succeptible bonds is reduced in the presence of calcium, whereas magnesium ions have no effect. It can be concluded from these results that the calcium binding centers are located in a more or less flexible zone of the molecule probably involving the C-terminal part of the Aalpha chain. And that the calcium divalent cation stabilizes a more compact structure of the fibrinogen molecule.     

A re-evaluation of some basic structural and functional properties of Pseudomonas cytochrome oxidase

Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.     

The relationship of 4-hydroxybenzoic acid to lysine and methionine formation in Escherichia coli

1. A multiple aromatic mutant, Escherichia coli 156:53D2, required 4-hydroxybenzoic acid for rapid aerobic growth on a number of carbon sources. 2. In the absence of 4-hydroxybenzoic acid aerobic growth was stimulated by a mixture of lysine and methionine and by succinate. The influence of the amino acids is attributed to a sparing of succinyl-CoA. 3. Low activities of both alpha-oxoglutarate dehydrogenase and fumarate reductase were found in organisms grown aerobically without 4-hydroxybenzoate and consequently both mechanisms known for the formation of succinate were impaired. 4. The low fumarate-reductase activity in these organisms was due to repression of enzyme synthesis by aeration and not to enzyme inactivation. In contrast lactate dehydrogenase and ethanol dehydrogenase were induced. This is interpreted as the appearance of alternative routes of NADH oxidation when electron transfer to oxygen is impaired. 5. The activities of the other tricarboxylic acid-cycle enzymes tested were little influenced by 4-hydroxybenzoate deficiency, although anaerobiosis resulted in a fall in activity.     

The M protein of SARS-CoV: basic structural and immunological properties

We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To inve     

Repression of the tyrosine, lysine, and methionine biosynthetic pathways in a hisT mutant of Salmonella typhimurium.

A comparison was made of the repressibility of certain enzymes in the tyrosine, methionine, and lysine biosynthetic pathways in wild-type Salmonella typhimurium and a hisT mutant. The results show that (i) tyrosine represses the synthesis of the tyrosine-sensitive 3-deoxy-D-arabino-heptulsonic acid 7-phosphate synthetase and the tyrosine aminotransferase to the same extent in a hisT mutant as in wild type and (ii) there is no detectable alteration in the extent to which methionine represses O-succinylhomoserine synthetase or in the extent to which lysine represses the lysine-sensitive beta-aspartokinase as a result of the hisT mutation.     

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.     

Seed-specific expression of a lysine rich protein sb401 gene significantly increases both lysine and total protein content in maize seeds

The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli using microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis and its expression was confirmed by Western blot analysis. Quantification of lysine and protein content in R1 maize seeds showed that, compared to the non-transgenic maize control, the lysine content increased by 16.1% to 54.8%, and total protein content increased by 11.6% to 39.0%. There was no visible morphological change in vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programmes aimed at improving the nutritional value of maize.     

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.