Modelling transmembrane region in G8 protein of Human and other

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International Journal of Computational Bioinformatics and In Silico Modeling Vol. 2, No. 4 (2013): 152-158 Research Article Open Access

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Modelling transmembrane region in G8 protein of Human and other organisms Mamta Sagar1*, Padma Saxena2, Neetesh Pandey1 and Dev Bukhsh Singh3 1 Department of Bioinformatics, University Institute of Engineering and Technology, Chhatrapati Shahu Ji Maharaj University, Kanpur-208024 2 Department of Zoology, Dayanand Anglo Vedic Post Graduate College, Chhatrapati Shahu Ji Maharaj University, Kanpur-208024 3 Department of Biotechnology, Institute of Biosciences and Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur-208024

*Corresponding author: Mamta Sagar; e-mail: [email protected]

ABSTRACT The G8 (for eight conserved glycine residues) domain act as half ABC trasporter is widely distributed, found in Homo sapiens. Uncharacterized Homolgs of this protein was obtained by sequence similarity search using BLAST and further analysed by multiple sequence alignment in Pan troglodytes, Nomascus leucogenys, Pongo abelii and Nomascus leucogenys. Phylogenetic results indicate that G8 protein of Homo sapiens is highly similar to G8 protein of Pan troglodytes, Nomascus leucogenys and closely related. Structure Modelling is done to predict structural similarities among transmembrane regions and its role as a transporter protein. Transmembrane prediction shows it is non-cytoplasmic in all organisms. Multiple sequence alignment analysis of transmembrane region indicates significant similarity except few amino acids, proline at 20th and histidine at 42nd position are present in human protein but leucine and arginine are present respectively in protein of Pan troglodyte. SSCH(R) LI motif is predicted among four sequences except in GIR3J8 protein of Nomascus leucogenys. Modeled structure of G8 proteins of Homo sapiens shows close similarities with its homologues in other three organisms. Transmembrane region LLWYLVFQYLLPGAGYILR using HMMPLOT is predicted in G1R3J8_NOMLE protein of Nomascus leucogenys but not in other sequences. Human G8 protein shows similarity to subunit an isoform 1 Q9Z1G4V-type proton ATPase (116 kDa) in Mus musculus which contain V type domain and Vacuolar proton translocating ATPase protein (Q93050) in Homo sapiens.

Keywords: G8 protein; ABC half transporter; Modelling; Pan troglodytes; Nomascus leucogenys; transmembrane

INTRODUCTION The G8 domain is widely distributed, being found in proteins from various animals (from Strongylocentrotus purpuratus to Homo sapiens), lower eukaryotes (such as Dictyostelium discoideum and Tetrahymena thermophila) and bacteria (such as the α-proteobacteria Nitrobacter hamburgensis, γ-proteobacterium Hahella chejuensis and green non-sulfur bacterium Chloroflexus aurantiacus) but absent in plants, viruses and archaea. Many G8-containing proteins are integral membrane proteins with signal peptides and/or transmembrane segments, and others lacking TM domain may be secreted POMGnT1 and TMEM2 proteins, with two well-conserved glycine residues Guo et al. and the PbH1

domain (SMART: SM00710,Parallel beta-helix repeats domain) [5 and 9]. G8 protein consists of approximately seventy five (75) amino acid residues. The secondary structure prediction of the G8 domain suggests that it contains ten (10) β-strands and one (01) helix. These strands are separated by conserved glycine residues and contain some conserved hydrophobic residues. The G8 domain is actually composed of five β-strand pairs Each repeat has a sequence resembling hX(0–3)hX(1–3)GX(1–11)hX(1–3)h, (X represents any amino acid residues and H represents hydrophobic residues). Most of the G8-containing proteins are predicted to be

Received: 22 May 2013 Accepted: 11 June 2013 Online: 01 July 2013


membrane-integral or secretary in nature. The G8 domain may be involved in extra cellular ligand binding and catalysis. It is an integral membrane receptor protein with extracellular protein-interaction sites and intracellular phosphorylation sites and may interact with extracellular protein–ligands and transduce intracellular signals to the nucleus [20]. Klein et al. described that G5 and G8 are both half-transporters associated to ABC proteins. These protein contain of ATPase catalytic domains positioned N-terminal to the transmembrane domain [11]. Venkateswaran et al. (2000) assessed G5 and G8 mediate the efficient excretion of neutral sterols. Lee et al. predicted ABCG5 (G5) and ABCG8 (G8) involve in translocation of a diverse substrates across cellular membranes [6, 8 and 13]. These are ABC transporter subfamilies in which protein contain one transmembrane domain one nucleotide binding fold. These ABC half-transporters make a functional complex. Sanchez-Fernandez et al. (2001); Yu et al. (2002) reported that G5 and G8 protein limit the absorption of sterols and promote cholesterol level. It has been observed PKHD1 may interact with extracellular protein–ligands and transduce intracellular signals to the nucleus [20]. Bergmann et al. detected autosomal recessive polycystic kidney disease (ARPKD) is associated with mutations in the polycystic kidney and hepatic disease (PKHD) gene on chromosome 6p12 [3]. He et al. predicted a novel protein domain G8 which contains five repeated β-strand pairs and is present in some disease-related proteins such as PKHD, KIAA1199, TMEM (Transmembrane) as well as other uncharacterized proteins [1&10]. Several other protein domains frequently co-occur in proteins with a G8 domain. Sabeva et al. reported G5 and G8 proteins are ABC half-transporters that dimerize within the endoplasmic reticulum. It mediates in cholesterol excretion into bile and also noted reduced biliary cholesterol excretion [17]. Prediction of G8 domain homologues is important for the research of the structure/function of related proteins and beneficial for the development of novel therapeutics. In this study G8 protein sequences is taken to characterize its function by searching protein homologs in other organism. Multiple sequence alignment is done using whole G8 protein and partial sequences of transmebrane regions predicted from Phobius tool in Homo sapience Pan troglodytes, Nomascus leucogenys, Pongo abelii. Modelling was done to compare transmemrane regions in G8 and its predicted homologues. MATERIALS AND METHODS Homology search and Phyologenetic analysis BLASTP is used to search homologous sequences [15] from NCBI using the representative sequences as a query. Multiple sequence alignment is done for homologous proein obtained from EBI BLAST to predict

the functions of uncharacterized genes. Phylogram tree shows how G8 protein of Homo sapiens is more closely related to proteins of other organisms; Pan troglodytes, Nomascus leucogenys and Pongo abelii while cladograms represent relationships between amino acid sequences. Transmembrane analysis The Phobius, a combined transmembrane protein topology and signal peptide predictor indicates that the N terminus of the mature protein must be on the non-cytoplasmic side of the membrane [14]. The predictor is based on a hidden Markov model [12]. A model shows different sequence regions of a signal peptide and the different regions of a transmembrane protein in a series of interconnected states. Homology search with transporter protein using TCDB BLAST (http://www.tcdb.org/progs/blast.php) TCDB is a database for transporter protein sequences and their classification. Blast is performed for G8 protein of Human with all proteins (http://www.tcdb.org/superfamily.php) present in database [18, 19]. RESULTS AND DISCUSSION Data base searching, protein sequence pattern analysis, conserved domain analysis, transmembrane helix prediction and structure modelling of protein were done to characterize the features of uncharacterized proteins. Data base searching The G8 protein is highly conserved among the different organisms such as Pan troglodytes, Nomascus leucogenys and Pongo abelii indicate good conservation at the amino acid level (Tables 1 and 2). G8 protein of Pan Troglodyte’s protein shows maximum similarity to G8 protein of Homo sapiens Protein Multiple sequence alignment of G8 protein and its homologues obtained by pairwise alignment using BLAST in different organisms shows that these are closely related (Figure 1 and 2).Two sequences TR:H2PIK8_PONAB and TR:G1R3J8_NOMLE showed only slight variations in G8 protein sequences. In Pongo abelii (TR:H2PIK8_PONAB) the protein sequence was 92 ٪ similar to G8 protein sequences while in the Nomascus leucogenys (TR:G1R3J8_NOMLE) the protein sequence was 83 ٪ similar to G8 protein sequences (Table -2). The similarity search matches G8 protein sequences in structure databases (Table - 2), using different BLAST programs, showed that there were highly significant (based on E-value and score) closely related homologues of the G8 protein. It indicates that Pan troglodyte’s protein show maximum similarity to G8_HUMAN (Q9UBA6) protein. The results of jalview indicates that H2QZW7_PANTR Pan troglodyte’s protein shows difference of two amino acid at position of 45th & 66th with G8_HUMAN (Q9UBA6) protein and closely related to human G8 protein as compare to other

Mamta Sagar et. al. / Int J Comput Bioinfo In Silico Model. 2013, 2(4): 152-158


species. Uncharacterized protein of Pan troglodytes shows the minimum distance with G8 of human. It is

predicted that both protein shows maximum similarity (Figure 4).

Table 1. BLAST result for G8 protein of human as a query sequence.

Align DB:ID Source Length Score Identities Positives E

1 SP:G8_HUMAN Protein G8 OS=Homo sapiens

GN=C6orf48 PE=2 SV=2 75 414 100.0 100.0 7.0E-39

2

TR:H2QZW7_PANTR

Uncharacterized protein OS=Pan

troglodytes GN=ENSG00000204387

PE=4 SV=1

75 396 97.0 97.0 9.0E-37

3

TR:G1R3J7_NOMLE

Uncharacterized protein (Fragment)

OS=Nomascus leucogenys

GN=ENSG00000204387 PE=4 SV=1

75 373 90.0 96.0 4.0E-34

4

TR:H2PIK8_PONAB

Uncharacterized protein (Fragment)

OS=Pongo abelii GN=C6orf48 PE=4

SV=1

73 353 92.0 93.0 9.0E-32

5

TR:G1R3J8_NOMLE

Uncharacterized protein

(Fragment)OS=Nomascus leucogenys

GN=ENSG00000204387 PE=4 SV=1

61 272 83.0 91.0 2.0E-22

Table 2. Similarity score of G8 proteins of Homo sapiens and its homologues in Pan troglodytes, Nomascus leucogenys and Pongo abelii.

SeqA Name Length SeqB Name Length Score

1 sp|Q9UBA6|G8_HUMAN 75 2 tr|H2QZW7|H2QZW7_PANTR 75 97.0

1 sp|Q9UBA6|G8_HUMAN 75 3 tr|G1R3J7|G1R3J7_NOMLE 75 90.0

1 sp|Q9UBA6|G8_HUMAN 75 4 tr|H2PIK8|H2PIK8_PONAB 73 94.0

1 sp|Q9UBA6|G8_HUMAN 75 5 tr|G1R3J8|G1R3J8_NOMLE 61 81.0

2 tr|H2QZW7|H2QZW7_PANTR 75 3 tr|G1R3J7|G1R3J7_NOMLE 75 90.0

2 tr|H2QZW7|H2QZW7_PANTR 75 4 tr|H2PIK8|H2PIK8_PONAB 73 94.0

2 tr|H2QZW7|H2QZW7_PANTR 75 5 tr|G1R3J8|G1R3J8_NOMLE 61 83.0

3 tr|G1R3J7|G1R3J7_NOMLE 75 4 tr|H2PIK8|H2PIK8_PONAB 73 93.0

3 tr|G1R3J7|G1R3J7_NOMLE 75 5 tr|G1R3J8|G1R3J8_NOMLE 61 90.0

4 tr|H2PIK8|H2PIK8_PONAB 73 5 tr|G1R3J8|G1R3J8_NOMLE 61 78.0

Figure 1. Jalview of multiple seuqence alignment shows amino acids in different colour, which is used to analyse variation among; G8 proteins of Homo sapiens and its homologues in Pan troglodytes, Nomascus leucogenys, Pongo abelii and Nomascus leucogenys.

Figure 2. Representing significant transmembrane region of G8 proteins of Homo sapiens (blue) and its homologues in Pan troglodyte (green), Nomascus leucogenys (red), Pongo abelii (green). Transmembrane region with high probability and less probability are shown in red and blue-green color respectively.



Multiple sequence alignment It is confirmed by similarity search result that not only G8 protein of Homo sapiens is similar to protein Q9UBA7 but also it is similar to uncharacterized protein of Pan troglodytes, Pongo abelii and Nomascus leucogenys, so it is revealed that all proteins are significantly related to each other and this provide fundamental basis for characterization of function of Half ABC transporter protein. No putative conserved domains have been predicted in these sequences. Multiple sequence alignment of transmembrane region Transmembrane region is predicted in G8 protein of Homo sapiens, Pan troglodytes, Nomascus leucogenys

and Pongo abelii. Multiple sequence alignment analysis of this region indicates that there is significant similarity between different organisms except few changes, in human protein proline at 20th position and histidine at 42nd position are present but leucine and arginine are present respectively in protein of Pan troglodytes. There are two gaps at the 8th and 29th position in Pongo abelii while proline and tyrosine are present on respectively in protein of other organisms. Nomascus leucogenys (GIR3J8) phenylalanine is present in place of threonine at 16th position and gap at 35th position. SSCH(R) LI motif present among four sequences except in GIR3J8 protein of Nomascus leucogenys

Figure 3a. Transmembrane prediction in G8 proten of Homo sapiens.

Figure 3b. Transmembrane prediction in G8 like protein of Nomascus leucogenys



Figure 4. Phylogenetic tree depict distances among G8 proteins of Homo sapiens and its homologues in Pan troglodytes, Nomascus leucogenys and Pongo abelii.

Modelling of G8 protein for Tran membrane helix prediction Result of present investigation indicates that G8 protein of Homo sapiens and the likely protein present in other organisms are non-cytoplasmic (Figure 3a and 3b) and contain a transmembrane region to be characterized as a half transporter. Out of five proteins two can be categorized as ABC transporter protein. This protein contain significant transmembrane region with high probability. Mostly G8-containing proteins are predicted to be membrane-integral or secreted [7]. Its domain may be involved in extra cellular ligand binding and catalysis. ABCG8 (G8) is member of the large family of ATP-binding cassette (ABC) transporters that facilitate translocation of a wide variety of substrates across cellular membranes [4 & 16]. The G subfamily is one ABC transporter protein subfamilies in which members contain one transmembrane domain and one nucleotide binding fold. Results of phobius (Figure 3a and 3b) reveal that functions of homologues proteins are similar to G8 protein. It is half-transporters and belonging to the G subfamily of ABC proteins. Based on the functions of G8-associated domains and proteins, it is sensible to predict that G8 may be involved in extra cellular ligand binding and catalysis process. Transmembrane regions are modeled and positions of transmembrane and signal peptide region are also located in modeled structure of G8 protein (Figure 5). Nucleotide binding domain consists of beta and extended region of secondary structures which contain signal peptide region (Figure 5a-5b) Transmembrane region from amino acid position 48th to 70th and 28th to 60th were predicted in G8 protein of Homo sapiens and Nomascus leucogenys. Signal peptide is predicted at start of sequence in Homo sapiens and Pan troglodytes. Amino acids in G8 like protein of Nomascus leucogenys vary with G8 protein of Human are found to be located in transmebrane region. Comparison of G8_Human with other Transmembrane (transporter protein) Transmembrane region was also predicted using HMMPLOT, this predicted motif LLWYLVFQYLLPGAGYILR in G1R3J8_NOMLE of Nomascus leucogenys. Transmembrane region using HMMPLOT is not predicted in any other sequences. Human G8 protein shows similarity to subunit a isoform 1 Q9Z1G4 V-type proton ATPase 116 kDa in Mus musculus which contain Vtype domain and Vacuolar proton translocating ATPase protein subunit (Q93050) in Homo sapiens.

Figure 5. Transmembrane region (cyan) in G8 protein of (a) Homo sapience and (b) Nomascus leucogenys and (c) Pan troglodytes amino acids (orange colour) are highlighted in G8 like protein of Nomascus leucogenys but not present in G8 of Homo sapiens. Signal peptide is shown in red colour in Pan troglodytes.



The V-type ATPases proteins are related to proton pumps that acidify intracellular compartments in eukaryotic cells and have important roles in membrane trafficking processes. The 116kDa subunit (subunit a) in the V-type ATPase is involve in proton transport. The subunit is a transmembrane glycoprotein with transmembrane helices, hydrophilic amino terminal and a hydrophobic carboxy terminal. It has roles in proton transport and assembly of the V-type ATPase complex encoded by VPH1 and STV1.

These results help to characterize function of ABC half transporter protein in human and other organism and predict the amino acid responsible for transportation of nutrients. These amino acids are VW, D, DS, R, G, S, PS+ L, T, PP which occur individually and in pair in G8 protein of Human. These amino acids are also present in V-type domain of proton ATPase protein in Mus musculus and vacuolar proton translocating ATPase protein subunit (Q93050) in Homo sapiens.

Figure 6. Sequence alignment with V-type proton ATPase subunit of Mus musculus and vacuolar proton translocation ATPase of Homo sapiens.

CONCLUSION The G8 domain is widely distributed in both animal and bacterial proteins including some hereditary disease related proteins PKHD1, KIAA1109 and TMEM2. Study provides structural and functional characterization of this protein. Sequence analysis indicate that G8 protein of Homo sapiens is closely related to Pan troglodytes, Nomascus leucogenys and Pongo abelii with similarity scores of 97, 90, 94 and 81% respectively. Phylogenetic results reveal that G8 protein of Human is closely similar to protein H2QZW7_PANTR of Pan troglodytes and G1R3J7_NOMLE of Nomascus leucogenys. Transmembrane regions are also similar in G8 protein of Homo and Pan troglodytes, Although significant transmembrane region is predicted in G8 protein Nomascus leucogenys. Results of modelling indicate that G8 protein of Homo sapiens and predicted homologues in these organisms are non-cytoplasmic. Structure modelling of G8 proteins of Homo sapiens revealed close similarities with its homologues in Pan troglodytes, Nomascus leucogenys and Pongo abelii, these predictions are also supported by transmembrane predictions. Signal peptide is also predicted in G8 protein of human and Pan troglodytes it

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Modelling transmembrane region in G8 protein of Human and other

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