Dogfish alpha-crystallin sequences. Comparison with small heat shock proteins and Schistosoma egg antigen

The amino acid sequences of the alpha-crystallin A and B chains of the dogfish, Squalus acanthias, have been determined. Comparison with alpha-crystallins from other species reveals that charged amino acid replacements have been strongly avoided in the evolution of this lens protein. The homology of alpha-crystallins with the small heat shock proteins is pronounced throughout the major part of the proteins, starting from the position of the first intron in the alpha-crystallin genes, but is also detectable in the amino-terminal sequences of human, Xenopus, and Drosophila small heat shock proteins. In addition, a remarkable short sequence similarity is present only in the amino termini of dogfish alpha B and Drosophila HSP22. The Schistosoma egg antigen p40 turns out to have a tandemly repeated region of homology with the common sequence domain of alpha-crystallins and small heat shock proteins. Comparison of hydropathy profiles indicates the conservation of conformation of the common domains in these three families of proteins. Construction of phylogenetic trees suggests that the alpha A and alpha B genes apparently originated from a single ancestral small heat shock protein gene and indicates that introns have been lost during the evolution of the heat shock protein genes.     

Structural organization of the bovine thyroglobulin gene and of its 5'-flanking region

The structural organization of the bovine thyroglobulin gene has been investigated by a combination of Southern genomic blotting and direct analysis of cloned gene fragments isolated from a chromosomal DNA library. The entire locus is spread over more than 200,000 base pairs which makes it one of the largest eukaryotic genes studies to date. The coding information is scattered into at least 42 exons, 34 of which have been precisely identified. A different evolutionary origin of the 5' and 3' regions of the gene is supported by the highly different proportion of exonic material they contain (12% and 3%, respectively) and by the existence of sequence homology between the 3' region of thyroglobulin and acetylcholinesterase. Detailed sequence analysis of the 5' region of the gene and its flanking segment demonstrated that a significant homology exists between bovine and human thyroglobulin sequences, except for the presence within the ruminant promoter region of a 220-base-pair sequence belonging to the bovine monomer repeated family.     

A critical evaluation of the hydropathy profile of membrane proteins

New membrane-preference scales are introduced for categories of membrane proteins with different functions. A statistical analysis is carried out with several scales to verify the relative accuracy in the prediction of the transmembrane segments of polytopic membrane proteins. The correlation between some of the scales most used and those calculated here provides criteria for selecting the most appropriate methods for a given type of protein. The parameters used in the evaluation of the hydropathy profiles have been carefully ascertained in order to develop a reliable methodology for hydropathy analysis. Finally, an integrated hydropathy analysis using different methods has been applied to several sequences of related proteins. The above analysis indicates that (a) microsomal cytochrome P450 contains only one hydrophobic region at the N-terminus that is consistently predicted to transverse the membrane: (b) only four of the six or seven putative transmembrane helices of cytochrome oxidase subunit III are predicted and correspond to helices I, III, V and VI of the previous nomenclature; (c) the product of the mitochondrial ATPase-6 gene (or the chloroplast ATPase-IV gene) of F0-F1-ATPase shows that helix IV is not consistently predicted to traverse the membrane, suggesting a four-helix model for this family of proteins.     

Identification of novel membrane proteins by searching for patterns in hydropathy profiles.

A technique has been developed to search a proteome database for new members of a functional class of membrane protein. It takes advantage of the highly conserved secondary structure of functionally related membrane proteins. Such proteins typically have the same number of transmembrane domains located at similar relative positions in their polypeptide sequence. This gives rise to a characteristic pattern of peaks in their hydropathy profiles. To conduct a search, each member of a polypeptide database is converted to a hydropathy profile, peaks are automatically detected, and the pattern of peaks is compared with a template. A template was designed for the acetylcholine (ACh) and glycine receptors of the cys-loop receptor superfamily. The key feature was a closely spaced triplet of hydropathy peaks bracketed by deep valleys. When applied to the human proteome the search procedure retrieved 153 profiles with a receptor-like triplet of peaks. The approach was highly selective with 70% of the retrieved profiles annotated as known or putative receptors. These included ACh, glycine, gamma-amino butyric acid and serotonin receptors, which are all related by sequence. However, ionotropic glutamate receptors, which have almost no sequence homology with ACh receptors, were also retrieved. Thus, the strategy can find members of a functional class that cannot be identified by sequence alignment. To demonstrate that the strategy can easily be extended to other membrane protein families, a template was developed for the neurotransmitter/Na+ symporter family, and similar results were obtained. This approach should prove a useful adjunct to sequence-based retrieval tools when searching for novel membrane proteins.     

An ABC transporter homologous to TAP proteins.

Polymerase chain reaction amplification of cDNA from rat intestine revealed the expression of a novel ABC transporter, TAPL (TAP-like). Subsequently, the protein sequence was deduced from the nucleotide sequence of cDNA carrying the entire coding region. TAPL is transcribed ubiquitously in various rat tissues. The protein, with 762 amino acid residues, has potential transmembrane domains, and an ATP-binding domain in its amino and carboxyl terminal regions, respectively, and is highly homologous to TAP1 and TAP2 (transporters associated with antigen presentation/processing): pairwise comparisons with TAPL demonstrated 39 and 41% of the residues are identical, respectively. These numerical values are essentially the same as that for TAP1 and TAP2 (39%), and the hydropathy profiles of TAPL, TAP1 and TAP2 are quite similar. The similarity among these three proteins suggests that they could be derived from a common ancestral gene. Furthermore, we found that there is a potential splicing isoform, sharing the amino terminal 720 amino acid residues of TAPL.     

Plasmodium falciparum: gene structure and hydropathy profile of the major merozoite surface antigen (gp195) of the Uganda-Palo Alto isolate

The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

The SAC domain-containing protein gene family in Arabidopsis

The SAC domain was first identified in the yeast (Saccharomyces cerevisiae) Sac1p phosphoinositide phosphatase protein and subsequently found in a number of proteins from yeast and animals. The SAC domain is approximately 400 amino acids in length and is characterized by seven conserved motifs. The SAC domains of several proteins have been recently demonstrated to possess phosphoinositide phosphatase activities. Sac1p has been shown to regulate the levels of various phosphoinositides in the phosphoinositide pool and affect diverse cellular functions such as actin cytoskeleton organization, Golgi function, and maintenance of vacuole morphology. The Arabidopsis genome contains a total of nine genes encoding SAC domain-containing proteins (AtSACs). The SAC domains of the AtSACs possess the conserved amino acid motifs that are believed to be important for the phosphoinositide phosphatase activities of yeast and animal SAC domain proteins. AtSACs can be divided into three subgroups based on their sequence similarities, hydropathy profiles, and phylogenetic relationship. Gene expression analysis demonstrated that the AtSAC genes exhibited differential expression patterns in different organs and, in particular, the AtSAC6 gene was predominantly expressed in flowers. Moreover, the expression of the AtSAC6 gene was highly induced by salinity. These results provide a foundation for future studies on the elucidation of the cellular functions of SAC domain-containing proteins in Arabidopsis.     

Presence of hormonogenic and repetitive domains in the first 930 amino acids of bovine thyroglobulin as deduced from the cDNA sequence

The sequence of the first 2831 nucleotides of bovine thyroglobulin mRNA has been determined from the analysis of a cDNA clone. Following a 41-nucleotide 5' untranslated sequence, a single open-reading frame encoding 930 amino acids was observed. This corresponds to the aminoterminal third of thyroglobulin, preceded by a putative signal peptide of 19 amino acids. The protein sequence was found to be essentially made of the sevenfold repetition of a 60-amino-acid-long building unit, interrupted at fixed positions by unrelated segments of variable length. The presence of an internal homology within the repetitive unit itself suggests that the 5' region of the thyroglobulin gene has evolved from the initial duplication of a relatively short sequence, followed by the serial duplication of the resulting unit. The tyrosine residue at position five has been assigned an important hormonogenic function [Mercken, L., Simons, M.-J. and Vassart, G. (1982) FEBS Lett. 149, 285-287]. This residue is flanked by sequence elements related to the repeated unit, suggesting that the hormonogenic domain evolved also from the basic ancestor sequence.     

Nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 12

The nucleotide (nt) sequence of a cloned DNA segment containing the early 2b region of the class A adenovirus Ad12 has been determined. When compared to the corresponding region of Ad2 or Ad7, there is a high degree of nt and predicted amino acid (aa) sequence homology within the r-strand regions that encode the preterminal protein and the viral DNA polymerase. A gene coding region comparable to the Mr 13,600 gene product found in Ad2 can be identified; this hypothetical gene product shares 30% aa homology with its Ad2 counterpart and has a very similar hydropathy profile.     

Membrane topology prediction by hydropathy profile alignment: membrane topology of the Na(+)-glutamate transporter GltS

Structural classification of families of membrane proteins by bioinformatics techniques has become a critical aspect of membrane protein research. We have proposed hydropathy profile alignment to identify structural homology between families of membrane proteins. Here, we demonstrate experimentally that two families of secondary transporters, the ESS and 2HCT families, indeed share similar folds. Members of the two families show highly similar hydropathy profiles but cannot be shown to be homologous by sequence similarity. A structural model was predicted for the ESS family transporters based upon an existing model of the 2HCT family transporters. In the model, the transporters fold into two domains containing five transmembrane segments and a reentrant or pore-loop each. The two pore-loops enter the membrane embedded part of the proteins from opposite sides of the membrane. The model was verified by accessibility studies of cysteine residues in single-Cys mutants of the Na+-glutamate transporter GltS of Escherichia coli, a member of the ESS family. Cysteine residues positioned in predicted periplasmic loops were accessible from the periplasm by a bulky, membrane-impermeable thiol reagent, while cysteine residues in cytoplasmic loops were not. Furthermore, two cysteine residues in the predicted pore-loop entering the membrane from the cytoplasmic side were shown to be accessible for small, membrane-impermeable thiol reagents from the periplasm, as was demonstrated before for the Na+-citrate transporter CitS of Klebsiella pneumoniae, a member of the 2HCT family. The data strongly suggests that GltS of the ESS family and CitS of the 2HCT family share the same fold as was predicted by comparing the averaged hydropathy profiles of the two families.     

Complete amino acid sequence of a basic 21-kDa protein from bovine brain cytosol

The complete amino acid sequence (186 amino acid residues) of a basic cytosolic protein from bovine brain has been determined. It was previously described as a phosphatidylethanolamine binding protein. Computer analyses have been used to calculate its hydropathy profile and to predict its secondary structure. Comparison with other proteins did not detect any significant sequence similarity, except for a short region which presents 53% sequence homology with bovine phosphatidylcholine transfer protein.     

The acetycholinesterase gene ofAnopheles stephensi

1. The acetylcholinesterase (AChE) gene from the important malaria vector Anopheles stephensi has been isolated by homology to the Drosophila acetylcholinesterase gene. 2. The complete sequence and intron-exon organization has been determined. The encoded protein has 69% identity to Drosophila AChE and 38 and 36% identity to Torpedo AChE and human butyrylcholinesterase, respectively.     

Thyroglobulin structure and function: recent advances

Thyroglobulin is a large-size iodoglycoprotein specific to thyroid tissue and is the substrate for the synthesis of thyroid hormones, thyroxine and 3,5,3'-triiodothyronine. Recent studies, which greatly benefited from recombinant DNA methodologies, improved the knowledge of several structural features of this dimeric protein and permitted insights into some structure-function relationships. Analysis-function of the primary structure of the human thyroglobulin monomer revealed several main characteristics: 1) 3 types of internal homologies; 2) extensive homology with the bovine thyroglobulin monomer and known partial sequences in the thyroglobulins of other mammalian species; 3) significant homologies with 2 other non-thyroid proteins (acetylcholinesterase and the invariant chain of the Ia class II histocompatibility antigen); 4) a terminal localization of the hormonogenic sites at both ends of the monomer. Current studies aim at determining conformational characteristics, understanding the molecular mechanisms of thyroid hormone formation and unraveling those interactions which in the thyroid cell and the thyroid follicle will permit this large pro-hormone to synthesize and release a few small thyroid hormone molecules. A more precise knowledge of this molecule in higher vertebrates and during evolution would impart valuable information concerning thyroid pathology, since thyroglobulin has been implicated in some genetic and in autoimmune thyroid diseases.     

The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases

The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli. The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226. Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E. coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size. Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar. The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene.