Identification and characterization of amphibian SLC26A5 using RNA-Seq

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Due to lacking of the 3D structure, most of the mechanism of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage. The knowledge of frog SLC26A5 is quite limited with only one species has been identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, the American bullfrog (Rana catesbeiana). In our study, the SLC26A5 gene of bullfrog has been mapped, sequenced and cloned successively using RNA-SEq. The comparative study revealed an alignment with nearly 40% identity among bullfrogs and mammalian species. The predicted 3D protein structure showed that the frog prestin possessed a transmembrane domain (TM) and a STAS domain similar to the mammalian prestin. The function of prestin crucially relies on its integration into the cell membrane. Such localization was observed when a prestin-EGFP fusion protein was expressed in HEK293T cells. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of frog’s inner ear and HEK293T cells transfected with this new coding gene. We observed that HEK293T cells expressing frog prestin showed electrophysiological features similar to that of hair cells from the amphibian’s inner ear. Conclusions We mapped and sequenced the SLC26A5 of the American bullfrog from its inner ear cDNA using RNA-SEq. The frog SLC26A5 cDNA was 2,292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. After isolating the prestin gene of the frog, we generated a stable cell line transfected with this new coding gene and found it possessing similar electrophysiological features as the hair cells from the frog’s auditory organ. Our experiment demonstrated that the new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line.

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Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽

10.1186/s12864-021-07798-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zhongying Wang ◽

Qixuan Wang ◽

Hao Wu ◽

Zhiwu Huang

Keyword(s):

Amino Acid ◽

Inner Ear ◽

Hair Cells ◽

Rana Catesbeiana ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Rna Seq ◽

American Bullfrog ◽

Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

Scientific Reports ◽

10.1038/s41598-021-96466-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Patamalai Boonserm ◽

Songchan Puthong ◽

Thanaporn Wichai ◽

Sajee Noitang ◽

Pongsak Khunrae ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

3D Structure ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Variable Regions ◽

Complementarity Determining Regions ◽

Crucial Part ◽

Major Amino Acid

AbstractIt is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

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PC8 [corrected], a new member of the convertase family

Biochemical Journal ◽

10.1042/bj3140727 ◽

1996 ◽

Vol 314 (3) ◽

pp. 727-731 ◽

Cited By ~ 63

Author(s):

Angela BRUZZANITI ◽

Katrina GOODGE ◽

Philippe JAY ◽

Sylvie A. TAVIAUX ◽

Mark H. C. Lam ◽

...

Keyword(s):

Amino Acid ◽

Transmembrane Domain ◽

Duplication Event ◽

Amino Acid Identity ◽

Chromosome 15 ◽

Rat Tissues ◽

Amino Acid Residues ◽

Degenerate Primers ◽

Chromosome 11Q23 ◽

Ancient Gene Duplication

A novel subtilisin-like protein, PC8, was identified by PCR using degenerate primers to conserved amino acid residues in the catalytic region of members of the prohormone convertase family. PC8 was predicted to be 785 residues long and was structurally related to the mammalian convertases furin, PACE4, PC1 and PC2, sharing more than 50% amino acid identity over the catalytic region with these family members. PC8 possessed the catalytically important Asp, His, Asn and Ser amino acids, the homo B domain of this family of enzymes and a C-terminal hydrophobic sequence indicative of a transmembrane domain. Structurally, PC8 is more related to furin and PACE4 than to PC1 or PC2. Like furin and PACE4, PC8 mRNA was found to be widely expressed; this is in contrast with PC1 and PC2, which have a restricted distribution. Two transcripts, of 4.5 and 3.5 kb, were detected in both human cell lines and rat tissues. Unlike furin and PACE4, both of which map to chromosome 15, PC8 maps to chromosome 11q23–11q24, suggesting that this gene may have resulted from an ancient gene duplication event from either furin or PACE4, or conversely that these genes arose from PC8.

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The HIV-1 Vpu transmembrane domain topology and formation of a hydrophobic interface with BST-2 are critical for Vpu-mediated BST-2 downregulation

10.1101/2020.06.28.176289 ◽

2020 ◽

Author(s):

Nabab Khan ◽

Siladitya Padhi ◽

Paresh Patel ◽

U. Deva Priyakumar ◽

Shahid Jameel

Keyword(s):

Amino Acid ◽

Transmembrane Domain ◽

Virus Production ◽

Cell Protein ◽

Amino Acid Residues ◽

Host Cell Protein ◽

Immunodeficiency Virus ◽

Molecular Modeling Studies ◽

Hiv 1 ◽

Hydrophobic Interface

AbstractViruses belonging to the M group of human immunodeficiency virus (HIV-1) are the most virulent among the four HIV-1 groups. One factor that distinguishes the M group HIV-1 from others is Vpu, a membrane localized accessory protein, which promotes the release of virions by neutralizing the antiviral host cell protein BST-2. To investigate if this activity is determined by the topology of Vpu or by conserved amino acid residues, we prepared chimeric forms of Vpu by replacing its transmembrane domain with those from its topological homologs. Although the chimeric Vpu proteins downregulated BST-2, these substantially reduced virus production as well. Molecular modeling studies on Vpu from different HIV-1 groups and the chimeric Vpu proteins showed that shape and the availability of a hydrophobic interface are more important for BST-2 antagonism than conservation of the amino acid sequence. Our data suggest that the HIV-1 Vpu-M protein has evolved topologically to interact with BST-2, and that the Vpu/BST-2 interface can be exploited as a target to limit HIV-1 replication.

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Inactivation of erythropoietin receptor function by point mutations in a region having homology with other cytokine receptors

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1788-1795.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1788-1795

Author(s):

O Miura ◽

J L Cleveland ◽

J N Ihle

Keyword(s):

Amino Acid ◽

Tyrosine Phosphorylation ◽

Transmembrane Domain ◽

Point Mutations ◽

Cell Receptor ◽

Erythropoietin Receptor ◽

Immediate Early ◽

Amino Acid Residues ◽

Dependent Cell ◽

Critical Residues

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a region, proximal to the transmembrane domain, that is essential for function and has homology with other members of the cytokine receptor family. To explore the functional significance of this region and to identify critical residues, we introduced several amino acid substitutions and examined their effects on erythropoietin-induced mitogenesis, tyrosine phosphorylation, and expression of immediate-early (c-fos, c-myc, and egr-1) and early (ornithine decarboxylase and T-cell receptor gamma) genes in interleukin-3-dependent cell lines. Amino acid substitution of W-282, which is strictly conserved at the middle portion of the homology region, completely abolished all the functions of the EpoR. Point mutation at L-306 or E-307, both of which are in a conserved LEVL motif, drastically impaired the function of the receptor in all assays. Other point mutations, introduced into less conserved amino acid residues, did not significantly impair the function of the receptor. These results demonstrate that conserved amino acid residues in this domain of the EpoR are required for mitogenesis, stimulation of tyrosine phosphorylation, and induction of immediate-early and early genes.

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Identification of betacellulin as a major peptide growth factor in milk: purification, characterization and molecular cloning of bovine betacellulin

Biochemical Journal ◽

10.1042/bj3440713 ◽

1999 ◽

Vol 344 (3) ◽

pp. 713-721 ◽

Cited By ~ 12

Author(s):

Andrew J. DUNBAR ◽

Ilka K. PRIEBE ◽

David A. BELFORD ◽

Chris GODDARD

Keyword(s):

Amino Acid ◽

Growth Factor ◽

Transmembrane Domain ◽

Signal Sequence ◽

Bovine Milk ◽

Gel Filtration ◽

Exchange Chromatography ◽

Amino Acid Residues ◽

Wide Range ◽

Rapid Amplification

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5ʹ and 3ʹ rapid amplification of cDNA ends (‘RACE’). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.

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A Specific Region of 37 Amino Acid Residues in the SPRY (B30.2) Domain of African Green Monkey TRIM5α Determines Species-Specific Restriction of Simian Immunodeficiency Virus SIVmac Infection

Journal of Virology ◽

10.1128/jvi.79.14.8870-8877.2005 ◽

2005 ◽

Vol 79 (14) ◽

pp. 8870-8877 ◽

Cited By ~ 86

Author(s):

Emi E. Nakayama ◽

Hiroyuki Miyoshi ◽

Yoshiyuki Nagai ◽

Tatsuo Shioda

Keyword(s):

Amino Acid ◽

Cell Line ◽

Specific Region ◽

Amino Acid Residues ◽

Immunodeficiency Virus ◽

Green Monkey ◽

Specific Restriction ◽

Species Specific ◽

Hiv 1 ◽

B30.2 Domain

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) efficiently enters cells of Old World monkeys but encounters a block before reverse transcription. This restriction is mediated by a dominant repressive factor. Recently, a member of the tripartite motif (TRIM) family proteins, TRIM5α, was identified as a blocking factor in a rhesus macaque cDNA library. Among Old World monkey cell lines, the African green monkey kidney cell line CV1 is highly resistant to not only HIV-1 but also simian immunodeficiency virus SIVmac infection. We analyzed TRIM5α of CV1 cells and HSC-F cells, a T-cell line from a cynomolgus monkey, and found that both CV1- and HSC-F-TRIM5αs could inhibit CD4-dependent HIV-1 infection, as well as vesicular stomatitis virus glycoprotein-mediated infection. CV1-TRIM5α could also inhibit SIVmac infection, whereas HSC-F-TRIM5α could not. In the SPRY (B30.2) domain of CV1-TRIM5α, there was a 20-amino-acid duplication that was not present in HSC-F-TRIM5α. A chimeric TRIM5α containing 37 amino acid residues from CV1-TRIM5α, which spanned the 20-amino-acid duplication, in the background of HSC-F-TRIM5α fully gained the ability to inhibit SIVmac infection. Conversely, the mutant CV1-TRIM5α lacking the 20-amino-acid duplication completely lost the ability to restrict SIVmac infection. These findings clearly indicated that a specific region of 37 amino acid residues in the SPRY domain of CV1-TRIM5α contained a determinant of species-specific restriction of SIVmac.

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Structural requirement of the calcium-channel subunit α2δ for gabapentin binding

Biochemical Journal ◽

10.1042/bj3420313 ◽

1999 ◽

Vol 342 (2) ◽

pp. 313-320 ◽

Cited By ~ 35

Author(s):

Minghan WANG ◽

James OFFORD ◽

Dale L. OXENDER ◽

Ti-Zhi SU

Keyword(s):

Amino Acid ◽

Structural Integrity ◽

Transmembrane Domain ◽

Anticonvulsant Drug ◽

Amino Acid Replacement ◽

Significant Loss ◽

Single Amino Acid ◽

Amino Acid Residues ◽

Structural Requirement ◽

High Binding Affinity

Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca2+-channel subunit α2δ. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain α2δ proteins were investigated. Removal of the disulphide bonds between the α2 and the δ subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed α2 protein remained membrane associated. However, α2 alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for δ, suggesting that both α2 and δ are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (δF) and 875-905 (δJ)] within the α2 subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (δD), 516-537 (δH) and 583-603 (δI)] within the α2 subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of α2, and regions H and I, between the putative splicing acceptor sites (Gln511 and Ser601), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg217 as critical for gabapentin binding.

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Analysis of the Transmembrane Domain of Influenza Virus Neuraminidase, a Type II Transmembrane Glycoprotein, for Apical Sorting and Raft Association

Journal of Virology ◽

10.1128/jvi.74.14.6538-6545.2000 ◽

2000 ◽

Vol 74 (14) ◽

pp. 6538-6545 ◽

Cited By ~ 88

Author(s):

Subrata Barman ◽

Debi P. Nayak

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Transmembrane Domain ◽

Mdck Cells ◽

Transmembrane Protein ◽

Apical Plasma Membrane ◽

Type Ii ◽

Amino Acid Residues ◽

Influenza Virus Neuraminidase ◽

Apical Sorting

ABSTRACT Influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. Previously, it was shown that the transmembrane domain (TMD) of NA provides a determinant(s) for apical sorting and raft association (A. Kundu, R. T. Avalos, C. M. Sanderson, and D. P. Nayak, J. Virol. 70:6508–6515, 1996). In this report, we have analyzed the sequences in the NA TMD involved in apical transport and raft association by making chimeric TMDs from NA and human transferring receptor (TR) TMDs and by mutating the NA TMD sequences. Our results show that the COOH-terminal half of the NA TMD (amino acids [aa] 19 to 35) was significantly involved in raft association, as determined by Triton X-100 (TX-100) resistance. However, in addition, the highly conserved residues at the extreme NH2 terminus of the NA TMD were also critical for TX-100 resistance. On the other hand, 19 residues (aa 9 to 27) at the NH2 terminus of the NA TMD were sufficient for apical sorting. Amino acid residues 14 to 18 and 27 to 31 had the least effect on apical transport, whereas mutations in the amino acid residues 11 to 13, 23 to 26, and 32 to 35 resulted in altered polarity for the mutant proteins. These results indicated that multiple regions in the NA TMD were involved in apical transport. Furthermore, these results support the idea that the signals for apical sorting and raft association, although residing in the NA TMD, are not identical and vary independently and that the NA TMD also possesses an apical determinant(s) which can interact with apical sorting machineries outside the lipid raft.

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