Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Evolution of Chitin-Binding Proteins in Invertebrates

Analysis of a group of invertebrate proteins, including chitinases and peritrophic matrix proteins, reveals the presence of chitin-binding domains that share significant amino acid sequence similarity. The data suggest that these domains evolved from a common ancestor which may be a protein containing a single chitin-binding domain. The duplication and transposition of this chitin-binding domain may have contributed to the functional diversification of chitin-binding proteins. Sequence comparisons indicated that invertebrate and plant chitin binding domains do not share significant amino acid sequence similarity, suggesting that they are not coancestral. However, both the invertebrate and the plant chitin-binding domains are cysteine-rich and have several highly conserved aromatic residues. In plants, cysteines have been elucidated in maintaining protein folding and aromatic amino acids in interacting with saccharides [Wright HT, Sanddrasegaram G, Wright CS (1991) J Mol Evol 33:283–294]. It is likely that these residues perform similar functions in invertebrates. We propose that the invertebrate and the plant chitin-binding domains share similar mechanisms for folding and saccharide binding and that they evolved by convergent evolution. Furthermore, we propose that the disulfide bonds and aromatic residues are hallmarks for saccharide-binding proteins. Received: 2 March 1998 / Accepted: 17 July 1998     

Relatedness and Phylogeny Within the Family of Periplasmic Chaperones Involved in the Assembly of Pili or Capsule-Like Structures of Gram-Negative Bacteria

The structure of a Salmonella enterica serovar typhi gene located within the fim gene cluster and encoding a putative periplasmic chaperone-like protein involved in the assembly of type 1 pili was determined. This gene, named fimC, has the ability to encode a 26-kDa polypeptide which is similar, at the sequence level, to the PapD periplasmic chaperonin mediating the assembly of P pili of Escherichia coli, as well as to other periplasmic chaperone-like proteins involved in the biogenesis of pili or capsule-like structures of various Gram-negative bacteria. A comprehensive search through the literature and sequence databases identified 31 (putative) bacterial proteins that can be included in this protein family on the basis of sequence similarity. Results of a multiple sequence comparison analysis showed that several residues, including most of those known to be critical in maintaining the three-dimensional structure of PapD, are either conserved or conservatively substituted in all these proteins, suggesting an overall similar folding for all of them. It was also evident that members of this family are clustered into different subfamilies according to structural and phyletic data. Received: 15 February 1996 / Accepted: 3 October 1996     

Positional Dependence, Cliques, and Predictive Motifs in the bHLH Protein Domain

Quantitative analyses were carried out on a large number of proteins that contain the highly conserved basic helix–loop–helix domain. Measures derived from information theory were used to examine the extent of conservation at amino acid sites within the bHLH domain as well as the extent of mutual information among sites within the domain. Using the Boltzmann entropy measure, we described the extent of amino acid conservation throughout the bHLH domain. We used position association (pa) statistics that reflect the joint probability of occurrence of events to estimate the ``mutual information content' among distinct amino acid sites. Further, we used pa statistics to estimate the extent of association in amino acid composition at each site in the domain and between amino acid composition and variables reflecting clade and group membership, loop length, and the presence of a leucine zipper. The pa values were also used to describe groups of amino acid sites called ``cliques' that were highly associated with each other. Finally, a predictive motif was constructed that accurately identifies bHLH domain-containing proteins that belong to Groups A and B. Received: 15 December 1997 / Accepted: 1 October 1998     

The Na-K-Cl Cotransporters

The Na-K-Cl cotransporters are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. The transport process mediated by Na-K-Cl cotransporters is characterized by electroneutrality (almost always with stoichiometry of 1Na:1K:2Cl) and inhibition by the loop diuretics bumetanide, benzmetanide, and furosemide. Presently, two distinct Na-K-Cl cotransporter isoforms have been identified by cDNA cloning and expression; genes encoding these two isoforms are located on different chromosomes and their gene products share approximately 60% amino acid sequence identity. The NKCC1 (CCC1, BSC2) isoform is present in a wide variety of tissues; most epithelia containing NKCC1 are secretory epithelia with the Na-K-Cl cotransporter localized to the basolateral membrane. By contrast, NKCC2 (CCC2, BSC1) is found only in the kidney, localized to the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop and of the macula densa. Mutations in the NKCC2 gene result in Bartter's syndrome, an inherited disease characterized by hypokalemic metabolic alkalosis, hypercalciuria, salt wasting, and volume depletion. The two Na-K-Cl cotransporter isoforms are also part of a superfamily of cation-chloride cotransporters, which includes electroneutral K-Cl and Na-Cl cotransporters. Na-K-Cl cotransporter activity is affected by a large variety of hormonal stimuli as well as by changes in cell volume; in many tissues this regulation (particularly of the NKCCl isoform) occurs through direct phosphorylation/dephosphorylation of the cotransport protein itself though the specific protein kinases involved remain unknown. An important regulator of cotransporter activity in secretory epithelia and other cells as well is intracellular [Cl] ([Cl]_i), with a reduction in [Cl]_i being the apparent means by which basolateral Na-K-Cl cotransport activity is increased and thus coordinated with that of stimulated apical Cl channels in actively secreting epithelia.     

The immunoglobulin superfamily: An insight on its tissular, species, and functional diversity

The immunoglobulin superfamily (IgSF) is a heterogenic group of proteins built on a common fold, called the Ig fold, which is a sandwich of two β sheets. Although members of the IgSF share a similar Ig fold, they differ in their tissue distribution, amino acid composition, and biological role. In this paper we report an up-to-date compilation of the IgSF where all known members of the IgSF are classified on the basis of their common functional role (immune system, antibiotic proteins, enzymes, cytokine receptors, etc.) and their distribution in tissue (neural system, extracellular matrix, tumor marker, muscular proteins, etc.), or in species (vertebrates, invertebrates, bacteria, viruses, fungi, and plants). The members of the family can contain one or many Ig domains, comprising two basic types: the constant domain (C), with seven strands, and the variable domain (V), with eight, nine, or ten strands. The different overviews of the IgSF led to the definition of new domain subtypes, mainly concerning the C type, based on the distribution of strands within the two sheets. The wide occurrence of the Ig fold and the much less conserved sequences could have developed from a common ancestral gene and/or from a convergent evolutionary process. Cell adhesion and pattern recognition seem to be the common feature running through the entire family. Received: 4 June 1997 / Accepted: 15 September 1997     

Na-K-Cl协同转运蛋白研究进展

Na-K-Cl协同转运蛋白是一类膜蛋白,负责转运Na、K、Cl离子进出上皮细胞与非上皮细胞。Na-K-Cl介导的转运过程是电中性的,多数情况下是1Na：1K：2C1（乌贼轴突中是2Na：1K：3C1）,其活性被布美他尼（bumetanide）和呋塞米（furosemide）所抑制。迄今为止,Na-K-Cl协同转运蛋白被鉴定出来两个同源异构体：NKCCl和NKCC2。NKCCl存在于多个组织中,合有NKCCl的上皮大多数属于分泌上皮,而且会有Na-K-Cl协同转运蛋白位于基底膜外侧;NKCC2只存在于肾脏,位于上皮细胞致密斑的顶膜上。Na-K-Cl协同转运蛋白的调控在不同的细胞和组织中是不同的。Na-K-Cl协同转运蛋白的活性会受激素刺激和细胞体积变化的影响;有些组织中,这种调控作用（尤其是NKCCl亚基）是通过特定的激酶使该转运蛋白自身发生氧化／硝化、磷酸化／去磷酸化来实现的;蛋白过表达在Na-K-Cl协同转运蛋白的激活中也起重要作用。     

The Antigen Receptor (NCCRP-1) on Catfish and Zebrafish Nonspecific Cytotoxic Cells Belongs to a New Gene Family Characterized by an F-Box-Associated Domain

The catfish nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. This report identifies and characterizes a zebrafish orthologue of the catfish NCCRP-1. The zebrafish NCCRP-1 cDNA contains an open reading frame that encodes a predicted protein of 237 amino acids with a MW of 27 kDa and a pI of 5.5. Sequence similarities comparisons show that the NCCRP-1 receptors from these two phylogenetically distant species share a high degree of identity. These results suggested that NCCRP-1 performs a crucial function in innate immunity in teleosts. Further, a zebrafish 17-mer peptide corresponding to the catfish NCCRP-1 antigen-binding domain inhibited (catfish) cytotoxicity toward conventional tumor target cells (HL-60). These data appeared to indicate that the zebrafish NCCRP-1 protein may function as an antigen recognition molecule and, as such, may participate in innate immunity in teleosts. A homology search of the zebrafish NCCRP-1 protein revealed that it shares a significant level of identity with another group of proteins belonging to an F-box subfamily. These proteins share an F-box domain in the N terminus (not present in NCCRP-1) and an extremely conserved C-terminal region that has been termed the F-box-associated domain (FBA). The FBA is currently of unknown function. A new gene family is proposed in this work, based on similarities in the FBA sequences with the catfish and zebrafish NCCRP-1 peptides. This new gene family includes several F-box domain-containing proteins and a predicted C. elegans protein. Received: 20 June 2001 / Accepted: 31 August 2001     

Na-K-Cl协同转运蛋白研究进展

Na-K-Cl协同转运蛋白是一类膜蛋白,负责转运Na、K、Cl离子进出上皮细胞与非上皮细胞。Na-K-Cl介导的转运过程是电中性的,多数情况下是1Na:1K:2Cl(乌贼轴突中是2Na:1 K:3Cl),其活性被布美他尼(bumetanide)和呋塞米(furosemide)所抑制。迄今为止,Na-K-Cl协同转运蛋白被鉴定出来两个同源异构体:NKCC1和NKCC2。NKCC1存在于多个组织中,含有NKCC1的上皮大多数属于分泌上皮,而且会有Na-K-Cl协同转运蛋白位于基底膜外侧;NKCC2只存在于肾脏,位于上皮细胞致密班的顶膜上。Na-K-Cl协同转运蛋白的调控在不同的细胞和组织中是不同的。Na-K-Cl协同转运蛋白的活性会受激素刺激和细胞体积变化的影响;有些组织中,这种调控作用(尤其是NKCC1亚基)是通过特定的激酶使该转运蛋白自身发生氧化/硝化、磷酸化/去磷酸化来实现的;蛋白过表达在Na-K-Cl协同转运蛋白的激活中也起重要作用。     

The Presence of GSI-Like Genes in Higher Plants: Support for the Paralogous Evolution of GSI and GSII Genes

Glutamine synthetase type I (GSI) genes have previously been described only in prokaryotes except that the fungus Emericella nidulans contains a gene (fluG) which encodes a protein with a large N-terminal domain linked to a C-terminal GSI-like domain. Eukaryotes generally contain the type II (GSII) genes which have been shown to occur also in some prokaryotes. The question of whether GSI and GSII genes are orthologues or paralogues remains a point of controversy. In this article we show that GSI-like genes are widespread in higher plants and have characterized one of the genes from the legume Medicago truncatula. This gene is part of a small gene family and is expressed in many organs of the plant. It encodes a protein similar in size and with between 36 and 46% amino acid sequence similarity to prokaryotic GS proteins used in the analyses, whereas it is larger and with less than 25% similarity to GSII proteins, including those from the same plant species. Phylogenetic analyses suggest that this protein is most similar to putative proteins encoded by expressed sequence tags of other higher plant species (including dicots and a monocot) and forms a cluster with FluG as the most divergent of the GSI sequences. The discovery of GSI-like genes in higher plants supports the paralogous evolution of GSI and GSII genes, which has implications for the use of GS in molecular studies on evolution. Received: 4 May 1999 / Accepted: 17 September 1999     

Accumulation of Species-Specific Amino Acid Replacements That Cause Loss of Particular Protein Functions in Buchnera, an Endocellular Bacterial Symbiont

Endosymbiotic bacteria live in animal cells and are transmitted vertically at the time of the host's reproduction. In view of their small and asexual populations with infrequent chances of recombination, these endocellular bacteria are expected to accumulate mildly deleterious mutations. Previous studies showed that the DNA sequences of these bacteria evolved faster than those of free-living bacteria. In this study, we compared all the ORFs of Buchnera, an endocellular bacterial symbiont of aphids, with those of 34 other prokaryotic organisms and estimated the effect of the accelerated evolution of Buchnera on the functions of its proteins. It was revealed that Buchnera proteins contain many mutations at the sites where sequences are conserved in their orthologues in many other organisms. In addition, amino acid replacements at the conserved sites are mostly changes to physicochemically different amino acids. These results suggest that functions and conformations of Buchnera proteins have been seriously impaired or strongly modified. Indeed, extensive loss of functional motifs was observed in some Buchnera proteins. In many Buchnera proteins mutations were not detected evenly throughout each molecule but tended to accumulate in some functional units, possibly leading to loss of specific functions. As Buchnera has an unusual and limited gene repertory, it is conceivable that the manner of interactions among its proteins has been changed, and thus, functional constraints over their amino acid residues have also been changed during evolution. This may account for the loss of some functional units only in the Buchnera proteins. We obtained evidence that amino acid replacements in Buchnera were not always deleterious, but neutral or, in some cases, even positively selected. Received: 14 December 2000 / Accepted: 12 March 2001     

New Glycoprotein-Associated Amino Acid Transporters

The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid substrate specificities, transport kinetics and localizations such as, for instance, y⁺LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein intolerance. The b^0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters (gpaAT family). Received: 20 July 1999/Revised: 7 September 1999     

Sequence Analyses and Phylogenetic Characterization of the ZIP Family of Metal Ion Transport Proteins

Several novel but similar heavy metal ion transporters, Zrt1, Zrt2, Zip1-4 and Irt1, have recently been characterized. Zrt1, Zrt2 and Zip1-4 are probably zinc transporters in Saccharomyces cerevisiae and Arabidopsis thaliana whereas Irt1 appears to play a role in iron uptake in A. thaliana. The family of proteins including these functionally characterized transporters has been designated the Zrt- and Irt-related protein (ZIP) family. In this report, ZIP family proteins in the current databases were identified and multiply aligned, and a phylogenetic tree for the family was constructed. A family specific signature sequence was derived, and the available sequences were analyzed for residues of potential functional significance. A fully conserved intramembranous histidyl residue, present within a putative amphipathic, α-helical, transmembrane spanning segment, was identified which may serve as a part of an intrachannel heavy metal ion binding site. The occurrence of a proposed extramembranal metal binding motif (H X H X H) was examined in order to evaluate its potential functional significance for various members of the family. The computational analyses reported in this topical review should serve as a guide to future researchers interested in the structure-function relationships of ZIP family proteins. Received: 31 March 1997/Revised: 14 May 1998     

Gene structure of a chlorophyll a/c-binding protein from a brown alga: Presence of an intron and phylogenetic implications

A Laminaria saccharina genomic library in the phage EMBL 4 was used to isolate and sequence a full-length gene encoding a fucoxanthin-chlorophyll a/c-binding protein. Contrary to diatom homologues, the coding sequence is interrupted by an intron of about 900 bp which is located in the middle of the transit peptide. The deduced amino acid sequence of the mature protein is very similar to those of related proteins from Macrocystis pyrifera (Laminariales) and, to a lesser extent, to those from diatoms and Chrysophyceae. Seven of the eight putative chlorophyll-binding amino acids determined in green plants are also present. Alignments of different sequences related to the light-harvesting proteins (LHC) demonstrate a structural similarity among the three transmembrane helices and suggest a unique ancestral helix preceded by two β-turns. The β-turns are conserved in front of the second helices of the chlorophyll a/c proteins more so than in chlorophyll a/b proteins. Phylogenetic trees generated from sequence data indicate that fucoxanthin-chlorophyll-binding proteins diverged prior to the separation of photosystem I and photosystem II LHC genes of green plants. Among the fucoxanthin-containing algae, LHC I or II families could not be distinguished at this time. Received: 14 February 1996 / Accepted: 4 April 1996     

Special evolutionary properties of genes encoding a protein with a simple amino acid repeat

We have examined the evolution of a gene, SM50, encoding a component of the spicule matrix, which plays an integral role in the formation of the echinoderm skeleton. This gene was originally characterized in Strongylocentrotus purpuratus and encodes an imperfect tandem repeat of six or seven amino acids. We have analyzed the sequence of this repeat in a number of sea urchin species and have determined that the repeat regions have undergone concerted evolution. There are differences in the repeat region between species, but the overall repeat structure is conserved, suggesting the repeat forms a structural domain important in biomineralization. The inherent conserved amino acid repeat structure promotes concerted evolution due to the high probability of misreplication and unequal crossing-over in the repeated segment of the gene. While there are constraints on the amino acids allowed in the repeat region, there are also variations, so that the sequences observed illustrate the balance between amino acid substitutions and concerted evolution. We have evidence that substitutions can alter the mechanisms of unequal crossing-over, altering the way concerted evolution occurs. The way in which concerted evolution occurred appears to be determined by the degree of sequence similarity between the repeats in a given gene, which influences how unequal crossing over may occur. We have mapped the differences in repeat regions on existing phylogenetic trees and indicate where concerted evolution has taken place. We also confirm an earlier report that Hemicentrotus pulcherrimus fits into the Strongylocentrotus genus and examine the evolution of the H. pulcherrimus SM50 repeat relative to other members of this genus. Received: 31 October 2000 / Accepted: 20 March 2001     

Acetylcholine Receptor Gating is Influenced by the Polarity of Amino Acids at Position 9′ in the M2 Domain

Ligand-gated ion channels contain a conserved leucine at position 9′ (L9′) in the M2 transmembrane domain. We used multiple substitutions at this position in the γ subunit of the mouse acetylcholine receptor (AChR) (γL9′) to examine the role of residue polarity at this position in the gating process at both the macroscopic and single-channel levels. The midpoint of the macroscopic dose-response relationship (EC₅₀) and the channel closing rate constant, α, decreased as the polarity of the residue at that position increased, suggesting a stabilization of the open state of the channel. Both parameters showed similar dependencies on the polarity of the substituted residue. These data support the notion that during AChR gating, the amino acid at the 9′ position moves into a polar environment, and that interactions between this residue and the polar environment determine the stability of the open state. Since this residue is conserved in all other members of the ligand-gated ion channel family, we suggest that a similar mechanism applies to the other members of the family. Received: 17 September 1999/Revised: 15 December 1999     

Permeability Increase Induced by Escherichia coli Hemolysin A in Human Macrophages is Due to the Formation of Ionic Pores: A Patch Clamp Characterization

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K⁺ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches. Received: 14 August 1995/Revised: 2 October 1995