Clathrin light chain: importance of the conserved carboxy terminal domain to function in living cells

Clathrin triskelions assemble into coats capable of packaging membrane and receptors for transport to intracellular destinations. A triskelion is formed from three heavy chains bound to three light chains. All clathrin light chains (clc) contain an acidic amino terminal domain, a central coiled segment, and a carboxy terminal domain conserved in amino acid sequence. To assess their functional contribution in vivo, we expressed tagged segments of the Dictyostelium clcA in clc-minus Dictyostelium (clc null) cells. We examined the ability of these clcA fragments to rescue clathrin phenotypic deficiencies, to cluster into punctae on membranes, and to bind to the heavy chain. When expressed in clc null cells, a clcA fragment containing the amino terminal domain and the central coiled domain bound heavy chain but was dispensable for clathrin function. Instead, the carboxy terminal domain of clcA was a critical determinant for association with punctae, for clathrin function and for robust binding to the heavy chain. A 70 amino acid carboxy terminal fragment was necessary and sufficient for full function, and for localization into punctae on intracellular membranes. A shorter 49 amino acid carboxy terminal fragment could distribute into punctae but failed to rescue developmental deficiencies. These results reveal the importance of the carboxy terminal domain of the light chain in vivo.     

Phospholamban: dissociation of the 22,000 molecular weight protein of cardiac sarcoplasmic reticulum into 11,000 and 5,500 molecular weight forms

Structural characterization of a 40 amino acid peptide with high intrinsic growth hormone releasing activity isolated from a human pancreatic tumor which had caused acromegaly was accomplished by gas phase sequence analyses of the intact peptide and its carboxy terminal cyanogen bromide digestion fragment. High pressure liquid chromatography of the native peptide and synthetic replicates showed that the molecule possessed a free acid rather than an amidated carboxy terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly- Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-OH using 1.8 nmoles of material. The structural identity of this material with a previously characterized fragment of a larger growth hormone releasing peptide isolated from a different human tumor is discussed.     

Primary structure of human plasma fibronectin--characterization of the 6,000 dalton C-terminal fragment containing the interchain disulfide bridges

The carboxy terminal fragment of human plasma fibronectin has been isolated after tryptic digestion and separation by DEAE-cellulose chromatography and gel filtration on Sephadex G-50. It has a molecular weight of 6,000 which changes to 3,000 after reduction indicating that the fragment is a dimer. We have determined the amino acid sequence of the 6kDa fragment and showed that it contains 26 residues including two half-cystines which form two interchain disulfide bridges. The 6kDa fragment is not phosphorylated as in bovine fibronectin although its amino acid sequence is identical to that reported for bovine plasma fibronectin. When compared to the sequence deduced from a rat cDNA, one amino acid substitution can be found. It appears that the carboxyl end of fibronectin is highly conserved among species.     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.     

The complete amino acid sequence of diphtheria toxin fragment B. Correlation with its lipid-binding properties

The complete amino acid sequence of fragment B from diphtheria toxin has been determined. The polypeptide chain was split with cyanogen bromide, o-iodosobenzoic acid, clostripain and trypsin; all amino acid sequence analyses were made by automated Edman degradation. Fragment B, which corresponds to the carboxy terminus of the toxin molecule, contains 342 amino acids and has an Mr of 37240. The proposed amino acid sequence fully confirms the structure recently deduced from the nucleotide sequence of the structural gene. The complete sequence is analyzed in relationship with the role of fragment B in the transfer of diphtheria toxin fragment A from the extracellular medium into the cell cytoplasm.     

Secretion of hemolysin of Aeromonas sobria as protoxin and contribution of the propeptide region removed from the protoxin to the proteolytic stability of the toxin

The sequence at the amino terminus region of the hemolysin ofAeromonas sobria is homologous with that of aerolysin of A. hydrophila. However, there is no homology between the two toxins in the sequence at the carboxy terminal region. It has been shown that aerolysin is secreted into culture supernatant as a protoxin. This proaerolysin is activated by the proteolytic removal of a carboxy terminal peptide. However, the role of the carboxy terminal region, which is removed in the activation process, has not been elucidated. In this study, we showed that hemolysin is also secreted as a protoxin into culture supernatant and that prohemolysin is cleaved by the protease of A. sobria between Ser-446 and Ala-447, resulting in the removal of a 42 amino acid peptide. The removal of the peptide converts the prohemolysin into active hemolysin. Subsequently, we mutated the hemolysin gene to delete the last several amino acid residues and expressed the genes in Escherichia coli, in order to examine the role of the carboxy terminal region of prohemolysin. The amounts of these mutant hemolysins accumulated in the periplasmic space of E. coli were very low compared with that of the wild-type. This observation indicated that the carboxy terminal region of prohemolysin contributes to the proteolytic stability of the toxin.     

Antithrombin Budapest 3 An antithrombin variant with reduced heparin affinity resulting from the substitution L99F

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, TC→ TC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT-binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution-induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.     

Protein structure prediction begins well but ends badly

The accurate prediction of protein structure, both secondary and tertiary, is an ongoing problem. Over the years, many approaches have been implemented and assessed. Most prediction algorithms start with the entire amino acid sequence and treat all residues in an identical fashion independent of sequence position. Here, we analyze blind prediction data to investigate whether predictive capability varies along the chain. Free modeling results from recent critical assessment of techniques for protein structure prediction (CASP) experiments are evaluated; as is the most up‐to‐date data from EVA, a fully automated blind test of secondary structure prediction servers. The results demonstrate that structure prediction accuracy is dependent on sequence position. Both secondary structure and tertiary structure predictions are more accurate in regions near the amino(N)‐terminus when compared with analogous regions near the carboxy(C)‐terminus. Eight of 10 secondary structure prediction algorithms assessed by EVA perform significantly better in regions at the N‐terminus. CASP data shows a similar bias, with N‐terminal fragments being predicted more accurately than fragments from the C‐terminus. Two analogous fragments are taken from each model, the N‐terminal fragment begins at the start of the most N‐terminal secondary structure element (SSE), whereas the C‐terminal fragment finishes at the end of the most C‐terminal SSE. Each fragment is locally superimposed onto its respective native fragment. The relative terminal prediction accuracy (RMSD) is calculated on an intramodel basis. At a fragment length of 20 residues, the N‐terminal fragment is predicted with greater accuracy in 79% of cases. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Mutation in the alpha 5(IV) collagen chain in juvenile-onset Alport syndrome without hearing loss or ocular lesions: detection by denaturing gradient gel electrophoresis of a PCR product.

A single base mutation was identified in the type IV collagen alpha 5 chain gene (COL4A5) of a Danish kindred with Alport syndrome. The 27-year-old male proband developed hematuria in childhood and terminal renal failure at the age of 25 years. He has no hearing loss or ocular lesions. Electron microscopy demonstrated splitting of the lamina densa of the glomerular basement membrane. The proband's mother has had persistent microscopic hematuria since the age of 40 years, but no other manifestations. Southern analysis of MspI-digested genomic DNA from the proband showed the absence of 1.3-kb and 0.9-kb fragments present in control DNA but the presence of a 2.2-kb variant fragment, indicating the loss of an MspI restriction site in the 3' end of the gene. The mother had all three fragments, indicating heterozygosity. PCR amplification of exon 14 (counted from the 3' end) and subsequent denaturing gradient gel electrophoresis analysis suggested a sequence variant in the proband and his mother. This was confirmed by sequencing of the PCR-amplified exon 14 region of the hemizygous proband, which demonstrated the base change G----A abolishing an MspI restriction site. Hybridization analysis with allele-specific probes confirmed the inheritance of the mutation with the phenotype. The mutation changed the GGC codon for glycine-1143 to GAC for aspartate. Substitution of glycine-1143, located in the collagenous domain of the alpha 5(IV) chain, for any other amino acid can be expected to interfere with the maintenance of the triple-helical conformation of the collagen molecule. This could, in turn, weaken the glomerular-basement-membrane framework and lead to increased permeability.     

Mutational analysis of the cleavage sequence of the human immunodeficiency virus type 1 envelope glycoprotein precursor gp160.

The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gp160, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp41. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gp120 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gp160 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. O. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gp120 amino acid 518, the terminal residue of gp120, abolishes both gp160 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gp160 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gp160 is required for activation of the HIV envelope fusion function.     

Structure of the plasmodium knowlesi gene coding for the circumsporozoite protein

The gene that codes for the surface antigen of Plasmodium knowlesi sporozoites (CS protein) is unsplit and present in the genome in only one copy. The CS protein, as deduced from DNA sequence analysis of the structural gene, has an unusual structure with the central 40% of the polypeptide chain present as 12 tandemly repeated amino acid peptide units flanked by regions of highly charged amino acids. The protein has an amino-terminal hydrophobic amino acid signal sequence and a hydrophobic carboxy-terminal anchor sequence. The coding sequence of the gene has an AT content of 53%, compared with 70% AT in the 5′ and 3′ flanking sequences, and is contained entirely within an 11 kb Eco RI genomic DNA fragment. This genomic fragment expresses the CS protein in E. coli, indicating that the parasite promoter and ribosome binding site signals can be recognized in E. coli.     

The second oncogene mil of avian retrovirus MH2 is related to the src gene family.

The nucleotide sequence of a PstI fragment prepared from a cloned MH2 virus genome, pMH2-Hd, has been deduced using chemical and enzymatic methods. This fragment, 1862 nucleotides in length, starts with the gag gene, encodes the v-mil sequence and stops within the v-myc gene. This sequence shows that the v-mil gene is fused to the gag gene giving rise to a fused polyprotein of 98 000 daltons: 515 amino acids at the amino terminus would correspond to p10, p19, p27 and part of p12 determinants, 347 amino acids at the carboxy terminus correspond to the v-mil specific sequence. The mil protein shares homology with a number of onc proteins such as src, fes, fms, mos, yes, fps and erbB, as well as with the catalytic chain of the cAMP-dependent protein kinase. This PstI fragment also encodes the beginning of the myc gene which was integrated in MH2 along with the 3' end of the preceding intron placing an acceptor splice site in front of the used open reading frame. As deduced from the sequence, the MH2 myc protein is not identical to the MC29 myc protein. It differs at its amino terminus, which contains little or no gag determinants, depending on the ATG used to initiate translation.     

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.     

Predicted Amino Acid Sequence of Bovine Tyrosine Hydroxylase and Its Similarity to Tyrosine Hydroxylases from Other Species

The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1-157) and 91% in the carboxy terminal (amino acids 158-491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.     

Characterization of the normal alpha 1-antitrypsin allele Vmunich: a variant associated with a unique protein isoelectric focusing pattern.

alpha 1-Antitrypsin (alpha 1AT), the major serum inhibitor of neutrophil elastase, is a highly polymorphic protein associated with isoelectric focusing (IEF) patterns typical for each variant. alpha 1AT Vmunich, a previously unreported normal alpha 1AT variant, has a unique IEF banding pattern in which the 7 and 8 alpha 1AT protein bands focus with the normal M-type 7 and 8 bands, despite the fact that the major fraction of the Vmunich protein focuses in the "V" region of the IEF gel. To characterize the molecular basis of this variant and its unique IEF pattern, DNA sequence analysis of the coding exons of the Vmunich alpha 1AT gene was carried out using the polymerase chain reaction. The Vmunich allele differed from the common normal M1(Val213) alpha 1AT allele by a single nucleotide substitution of cytosine for adenosine, with the resultant amino acid change Asp2 GAT----Ala GCT. Inheritance of the allele was confirmed by family analysis using allele-specific amplification with the polymerase chain reaction. The Asp2----Ala mutation explains the cathodal position of the Vmunich protein on IEF, as there is a substitution of a negatively charged amino acid by a neutral one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a functional domain of human calpastatin

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.     

Apolipoprotein E-4Philadelphia (Glu13----Lys,Arg145----Cys). Homozygosity for two rare point mutations in the apolipoprotein E gene combined with severe type III hyperlipoproteinemia.

The molecular defect in a 24-year-old white female with severe type III hyperlipoproteinemia has been elucidated. The patient's apolipoprotein (apo) E migrated in the apoE-4 position on isoelectric focusing gels. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apoE-4 variant had a smaller apparent molecular weight than apoE-4(Cys112----Arg). Sequence analysis of DNA amplified with the polymerase chain reaction revealed two nucleotide substitutions in the proband's apoE gene. A C to T mutation converted arginine (CGT) at position 145 of the mature protein to cysteine (TGT) thus creating the apoE-2 variant. A second G to A substitution at amino acid 13 led to the exchange of lysine (AAG) for glutamic acid (GAG), thereby adding 2 positive charge units to the protein and producing the apoE-5 variant. Computer analysis of the apoE-4Philadelphia gene revealed that the G to A mutation in exon 3 resulted in the loss of an AvaI restriction enzyme site. The second mutation, a C to T substitution in the fourth exon of the apoE gene, eliminated a cleavage site for the enzyme BbvI. Using these restriction fragment length polymorphisms as well as DNA sequence analysis we have demonstrated that the patient is homozygous for both point mutations in the apoE gene.     

Isoforms of soybean seed oil body membrane protein 24 kDa oleosin are encoded by closely related cDNAs

We have characterized two cDNA clones for 24 kDa soybean oleosin, the seed oil body membrane protein. Differences in the predicted amino acid sequences of the two clones and the presence of a doublet on immunoblots indicate that 24 kDa oleosin exists in at least two isoforms in soybean. The predicted amino acid sequence also contains a unique carboxy terminal region that is dominated by a series of different tandem amino acid repeats.