In Silico investigation for hemoglobin characterizations leads to new aspects in susceptibility to glycosylation  

Reza Talebi1 , Ahmad Ahmadi1 , Fazlolah Afraz2 , Seyed Zeyaedin Mirhoseini3
1. Department of Animal Genetics, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran
2. Department of Animal Genetics, Agricultural Biotechnology Research Institute of Iran, Rasht, Iran
3. Department of Animal Genetics, Faculty of Agriculture, University of Guilan, Rasht, Iran
Author    Correspondence author
Computational Molecular Biology, 2015, Vol. 5, No. 1   doi: 10.5376/cmb.2015.05.0001
Received: 10 Dec., 2014    Accepted: 25 Dec., 2014    Published: 29 Dec., 2014
© 2015 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Talebi et al., 2015, In Silico investigation for hemoglobin characterizations leads to new aspects in susceptibility to glycosylation, Computational Molecular Biology, Vol.5, No.1, 1-13 (doi: 10.5376/cmb.2015.05.0001)

Abstract

In silico analysis were performed between some species for hemoglobin characterizations specially resistivity to glycosylation. Multiple alignments of both hemoglobin subunits were revealed L-K-V-K-E-G-E-A-L-R-P-T-F-F-D-L-S-A-V- K-H-G-K-V-H-D-L-S-L-H-K-L-V-D-P-N-F-L-L-L-A-F-T-P-A-K-V-L-Y conserved in camel, human, cow and horse entirely. Hemoglobin sequences are well conserved in evolution and between species. Camelus families HBA were in same cluster (91% bootstrapping). However for HBB they were in same cluster (95% bootstrapping). Camel hemoglobin appeared to be more basic than either that of other species. Based on isoelectric points, camel HBB has high mobility than HBA in electrophoresis. The charged and hydrophilic amino acids of hemoglobin in camel were more than human. The non-enzymatic binding of glucose to the protein or HbA1c is revealed both subunits hemoglobin in human is resistance to N-glycosylation but camel HBB is only resistant to O-glycosylation. HBB is less susceptible to glycosylation than HBA. Also HBB has critical roles in camel for instance resistivity to glycosylation and diabetes subsequently.

Abbreviations: HB, Hemoglobin; HBA, α-chain of hemoglobin; HBB, β-chain of hemoglobin; GHB, glycosylated hemoglobin; AA, Amino Acid; NA, nucleic acid; Vs., versus; pI, Isoelectric Point.

Keywords
In silico; Multiple alignments; Hemoglobin; Glycosylation; HbA1c.

Globins are unique molecules that could transport oxygen through body tissues. Hemoglobin and myoglobin are two kinds of globins that are respectively responsible in blood and muscle. They are found in all vertebrates and some kinds of invertebrates. Hemoglobin structure in vertebrates shows the typical globin fold accommodating a heme group which reversibly binds oxygen to its central ferrous atom compound. Adult hemoglobin is a [α(2):β(2)] tetrameric hemoprotein with molecular mass of each chain is 15 kDa which is found in erythrocytes, where it is responsible for binding oxygen in the lung and transporting the bound oxygen throughout the body where it is used in aerobic metabolic pathways (Dickerson and Geiss, 1983; Paoli and Nagai, 2004; Pairet and Jaenicke, 2010) The shapes of erythrocytes in the mammalian(s) except of camelidae family are spherical and concave, but in the camelidae, are oval form and nucleus less (Goniakowska-Witalinska and Witalinski, 1976; Azwai et al., 2007). The erythrocytes of camelidae are highly resistant against osmotic hemolysis, so are able to expand up to 240% of their original volume without rupturing in hypotonic solutions (Oyewale et al., 2011).

Glycosylated hemoglobin (GHB) or Maillard reaction is formed by a non-enzymatic interaction between glucose and N-terminal valine and lysine residue of the β-chain of HB, maybe should be causes rise to HbA1c- a type of HB commonly associated with diabetes (Rohlfing et al., 2002; Wautier and Schmidt, 2004; Soranzo, 2011). As HbA1c is resided for long term and has’nt decomposed, HbA1c concentrations have been used for decades to assess the effects of long-term glycemic control in diabetic patients.10 Formation of GHB is irreversible and its level in the erythrocytes depends on the blood glucose concentration. Measurement of GHB, which was first introduced in the 1970's, provides an index of long term glycemic control, which has been proven to evoke changes in diabetes treatment resulting in improved metabolic control. An HbA1c extent directly relates to the blood glucose levels in human, and maybe so in other animals (Nathan et al., 2008; Shahbazkia et al., 2010; Bazzi et al., 2013) Based on the correlation between the AA and glucose, the process of glycosylation in eukaryotes could be categorized into five types: N-linked, O-linked, C-linked, P-linked and G-linked which N- and O-linked are most common glycosylation forms (Chauhan et al., 2013).
Blood glucose concentration ratio in camel and cow is about (9.7±2.8 mM) (at high level) and (5.7±0.73 mM) (in lowest level). High level of blood glucose in camels may be caused by their strong capacity for insulin resistance (Jirimutu et al., 2012; Bazzi et al., 2013). Level of glycosylated hemoglobin (GHB) is 3.4± 0.23% and 3.2± 0.11% in camel and cattle, respectively. The low glycation of camel Hb at higher glucose concentrations suggests that certain factors protect the Hb from glycation at high glucose concentrations (Bazzi et al., 2013). According to Oyewale et al. (2011) Camelus dromedarius hemoglobin provides an interesting case study of adaptation to life in deserts at extremely high temperatures. As well as camel Hb also exhibited higher electrophoretic mobility than normal hemoglobin in human or cow (Oyewale et al., 2011).
Lot numbers of researches have been carried out on the erythrocytes and hemoglobin of camel species (Lin et al., 1976; Braunitzer et al., 1980; Farooq et al., 2011; Oyewale et al., 2011). Oyewale et al (2011) had compared erythrocytes fragility between camel (Camelus dromedarius) and donkey (Equus asinus) in different environmental situation. They had suggested that variation in the temperature and pI of the erythrocyte environment beside duration of blood storage may each play a significant role in the osmotic behavior of camel and donkey erythrocytes (Oyewale et al., 2011). Bazzi et al (2013) had revealed high blood glucose concentration (9.7±2.8 mM) and low level of glycated-Hb (3.4± 0.23%) in camel but cow blood samples did not show sufficient variations in glucose concentrations (5.7±0.73 mM) or glycated-Hb levels (3.2± 0.11%) (Bazzi et al., 2013). They had declared low glycation of camel Hb at higher glucose concentrations revealed certain factors which protect the Hb from glycation at high glucose concentrations. Level of glycated-Hb may not only just reflect dependence on blood glucose level, erythrocyte life span and permeability of erythrocyte membrane, but also shows on food regime (Ardia, 2006) Borai et al. (2011) had suggested HbA1c can be used as a simple and reliable marker of insulin resistance in normal adult’s glucose tolerance with relatively high insulin sensitivity (Borai et al., 2011). According to previous researches on the role of hemoglobin in diabetes, we decided to study on structure, chemical features and compositions of hemoglobin in the Camelus dromedaries, Camelus bactrianus,Camelus ferus, Bos taurus, Homo sapiens and Equus caballus. Despite the fact that camels have higher blood glucose levels than human, the extent of glycosylation is extremely less in camel’s blood than in ours. Therefore, DNA and protein sequences of HB subunits were compared between mentioned species to study on resistivity of glycosylation and to diabetes subsequently.
1 Material and methods
1.1 Data collection
The AA and nucleic acid (NA) sequences of HB (alpha and beta subunits) were obtained for Camelus dromedaries, Camelus bactrianus, Camelus ferus, Bos taurus, Homo sapiens and Equus caballus from biological database such as Uniprot and KEGG (Table 1).


Table 1 α and ß chains of hemoglobin are obtained from accessing biological database. Protein names, Access number and GeneBank following is shown


1.2 In silico analysis
Sequences had converted to amino acid (*.pro) by complex of Lasergene softwares. As well as mentioned files were altered to FASTA form (*.fas), for which this form is common for analysis of genomic data’s. We used ClustalW to perform multiple alignments for proteins in each species by MEGA 5.01 (Tamura et al., 2011) and CLC Genomics Workbench 7.5. The evolutionary history was inferred by using the UPGMA based on Kimura protein distance measure. UPGMA assumes a constant rate of evolution.
1.3 Protein composition
Predicted structural classes of the whole protein were used by Alpha Deléage & Roux Modification of Nishikawa & Ooi 1987 (Deléage and Roux, 1987). Protein structure, titration curves, AAs composition and frequency of each protein were performed by the Protean (DNASTAR Inc., Madison, WI. USA).
1.4 Prediction of protein structure
We predict tertiary structure of hemoglobin subunits based on homology-modelling using of SWISS-MODEL available in the ExPASy website (http://swissmodel.expasy.org/). Which, its purpose is to make Protein Modelling accessible to all biochemists and molecular biologists all around the world.
1.5 Prediction of N- and O-Glycosylation sites
In this study we utilized a new webserver GlycoEP (http://www.imtech.res.in/raghava/glycoep/submit.html) for more accurate prediction of N-linked, O-linked and C-linked glycosylation sites of HBA and HBB or non-enzymatic binding of glucose to the protein (as in the case of HbA1c) in camel species and human.
1.6 Comparative modeling
In order to compute comparative modeling, we used some criterions such as homology, conserved, consensus and E-value by BlastP amongst mentioned species and compare the NCBI HomoloGene database to assign them to the gene families. Eventually, PSI-BLAST (Position-Specific Iterated BLAST) was done in order discovering of HomoloGene between camel hemoglobin and various species of protein in the chosen database (Altschul et al., 1997).
2 Results and Discussion
2.1 Multiple alignments of hemoglobin subunits:
The multiple alignments were revealed Leu, Lys, Val, Lys, Glu, Gly, Glu, Ala, Leu, Arg, Pro, Thr, Phe, Phe, Asp, Leu, Ser, Ala, Val, Lys, His, Gly, Lys, Val, His, Asp, Leu, Ser, Leu, His, Lys, Leu, Val, Asp, Pro, Asn, Phe, Leu, Leu, Leu, Ala, Phe, Thr, Pro, Ala, Lys, Val, Leu and Tyr that these are conserved entirely in all hemoglobin’s subunits (Figure 1). Due to the conserved sequences it could be conjectured that the least mutation had occurred in these kinds of AA for mentioned species or some aa because had eliminated by mutations that resided these conserved sequences for hemoglobin subsequently. On the base of conserved sequence Leu has major contribution than others, so that according to the Binder et al., 2013, beneficial effects of leucine on intestinal gluconeogenesis and islets of Langerhans’s physiology might help prevent diabetes type II (Binder et al., 2013). Meanwhile, on the base of Figure 1 a lot of AAs are conserved for alpha and beta subunit of hemoglobin separately in mentioned species, so this indicates that the least mutation has occurred in hemoglobin structure after divergence of species. So we can conclude that the structure of the hemoglobin protected against the mutagenic agents, thus the most unique efficiencies of hemoglobin in the camels or other species is related to structure, composition and features of alpha HBA and HBB due to the fact that alpha and beta subunit is highly conserved in camel, cow, horse and human. As well as illustrated on Figure 3 each chains of hemoglobin only have one domain (domain 1) in the present study.


Figure 1 Multiple sequence alignment of HB subunits. 1A) α and ß chains of hemoglobin were aligned between human, domestic one and two-humped camel, wild two humped camel, cow and horse. 1B) Α chain of hemoglobin was aligned between mentioned species. 1C) B chain of hemoglobin was aligned between mentioned species. Identical gaps and conserved sequences are indicated by dashes (-) and pink columns respectively


The most frequencies of AAs with consensus sequences belonged to L-K-V-K-E-G-E-A-L-R-P-T-F- F-D-L-S-V-K-H-G-K-V-H-L-D-L-L-S-L-H-K-L-V-D-P-N-F-L-L-L-A-F-T-P-A-K-V-L-Y (Figure 1) that these are most common modes could be possible. Based on the consensus sequence can identified the nucleotides that are less affected by the mutations. For instance according to Figure 1, in the part of alpha subunit of hemoglobin in cow the AA (amino acids) sequences of A-A-G-A-W-V-S-G-A-A-A were substituted by S-K-T-T-F-I-G-A-K-G-D in the one humped camel respectively. As well as, in the part of alpha subunit of hemoglobin in the Camelus bactrianus and Camelus dromedarius, amino acids sequences of S-K-T-F-I-G-A-G were substituted by P-A-A-W-V-A-G-S in Homo sapiens respectively, which is also showed in the results of the phylogenetic diagram (Figure 2). Substitution of amino acid was not found between Camelus family for both subunits but it was obviously found for other species (Figure 1). So it maybe relates to hemoglobin sequences are well conserved in evolution and between species. In some parts of ß-chain sequences in the Camelus family (Camelus bactrianus, Camelus dromedarius and Camelus ferus) the AA of H, D, N, H, G, S, K, V and D were substituted by Q, E, A, L, A, D, N, E and E in Equus caballus respectively or the AA of S, G, D, N, H, G, S, K and R in Camelus family were substituted by T, P, E, S, T, A, G, N and Q in Homo sapiens respectively. So, differentiation of amino acid in the species occurred during mutation into same polypeptide which is major factor concerning to hemoglobin efficiency in creatures. So, mutations have played an important or unique role in causing animals adapting to different environmental situations. Lin et al., 1976 are declared that between adult camel hemoglobin and adult human hemoglobin six amino acid differences were found in the N-terminal 20 amino acid residues of the α-chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the ß chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at α5 Ala → Lys, and ß19 Asn → Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no meaningful differences (Lin et al., 1976). Pieragostini et al., 2010 presented an adaptive significance of the hemoglobin variants with hematological patterns in ruminant breeds (Pieragostini et al., 2010). Braunitzer et al., 1980 declared five and two AAs (amino acids) in HBA and HBB of Camelus ferus are exchange versus lama respectively. As well as they deduced the AAs sequence of Camelus ferus and dromedarius is identical (Braunitzer et al., 1980). However, we hypothesized that substitution of AAs into polypeptide structure by protein engineering or cloning is an appropriate approach to how increasing resistivity of hemoglobin to glycosylation and subsequently to diabetes mellitus.
2.2 Phylogenetic analysis
As shown in circular cladogram Figure 2 HbA in Camelus bactrianus and Camelus ferus were in the same sub cluster with 100% bootstrapping near alpha subunit of Camelus dromedarius. It also shown for HBB in Camelus family (Figure 2). As phylogenetic distance of Camelus bactrianus and dromedariusα-chains versus Homo sapiens were more than Bos taurus and Equus caballus (Figure 2). Phylogenetic tree revealed ß-chain (HBB) of Camelus bactrianus, Camelus dromedarius and Camelus ferus were in the same sub cluster with 95% bootstrapping and were 55% bootstrapping close to HBB of Equus caballus. The based on circular cladogram Figure 2 HBA in Equus cabalus and Bos tauurus were in the same sub cluster with 39% bootstrapping but far from HBA Homo sapiens slightly. Phylogenetic analysis had demonstrated, every hemoglobin subunits are conserved in species and it depends on particular function and performance of protein.


Figure 2 Circular cladogram by UPGMA. The evolutionary history was inferred by using the UPGMA method based on the JTT matrix-based model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Initial tree(s) for the heuristic search were obtained automatically as follows. The analysis involved 12 amino acid sequences


2.3 Whole protein composition
Whole protein composition of HBA and HBB were the same in the Camelus family but and was different from other species entirely (Table 2). The based on Table 2, isoelectric point (pI) of hemoglobin in Camelus appeared to be more than either that of Homo sapiens, Bos tauurus and Ecuus cabalus (Table 2). In fact pI for hemoglobin subunits in camel is basic rather than human. Also hemoglobin subunits in camel contain more positively and negatively charged amino acids than either Homo sapiens. The basic effects of pI in camels HBB is more than HBA (Table 2), thus we imagine it’s related to high mobility of camel HBB than HBA in electrophoresis and resistance to glycosylation subsequently. However we considered isoelectric point in HBB for Camelus family were more than human, cow and less than horse slightly (Table 2). In other word, ß-chain (HBB) in camel and human has basic and neutral characteristics or that it may influence glycosylation. It affects mobility of protein during electrophoresis, so camel and human HBB respectively have high and low mobility during electrophoresis. As well as Bazzi et al., 2013 declared camel Hemoglobin has higher electrophoretic mobility than normal hemoglobin of human or cow (Bazzi et al., 2013). The based on Table 2, pI for α-chain of hemglobin (HBA) in Camelus bactrianus and Camelus dromedarius were the same (9.39) more than Camelus ferus (9.33) slightly. Camel pI for both hemoglobin subunits were more than Homo sapiens (Table 2). According to the findings and Table 2, we noticed that a notable difference in whole protein composition of HBA between human and camel does not exist. So we envision that other scenarios are affected to by minimal glycosylation of camel hemoglobin - for instance camel erythrocytes may restrict glucose transport more than human-. Oyewale et al., 2011 were found that camel erythrocytes are more resistant to hemolysis (or less osmotically fragile) than donkey erythrocytes (Oyewale et al., 2011). In this study, as illustrated in Figure 2, the Camelus family hemoglobin has higher proportion of hydrophilic amino acids (polar) compared with human (for instance 20.42 and 21.28 for HBA between human and camel respectively), and this gives the camel hemoglobin its hydrophilic character Charged AAs (amino acids) for HBA and HBB in Camelus family are more than human (Table 2). It could also be possible that the redox state of camel blood or the internal environment of erythrocytes as variations of blood pH may contribute to the reduction of HbA1c levels, as the reseach of Bazzi et al., 2013. As well as Lin et al., 1976 are stated Camelus dromedarius hemoglobin possesses more positive charges on paper electrophoresis than does human HBA, and showed that been consist of only a single component (Lin et al., 1976). Some researchers are declared that erythrocytes of camels is more resistant to hemolysis in comparison with cattle, sheep, goats, pigs, mice, man and other animals (Al-Qarawi et al., 2004; Oyewale et al., 2011). As hemoglobin from Camelus dromedarius provides an interesting case of adaptation to life condition in deserts at extremely high temperatures (Balasubramanian et al., 2009) The proportion of water in the camel erythrocytes that is osmotically non-removable is almost three times greater than in human, which seems leading to extremely good resistance of camel erythrocytes to osmotic change (Bognera et al., 2005). As influence of food on glycosylation of hemoglobin (GHB) in the birds is demonstrated (Miksik and Hodny, 1992).


Table 2 Composition of hemoglobin. Predicted structural class of the whole protein: Alpha Deleage & Roux Modification of Nishikawa & Ooi 1987


2.4 Homology measurement
Based on information demonstrated in Table 3, similarity of HBA and HBB between Camelus bactrianus and Camelus dromedarius was 100%, as 100% query coverage (Table 3). However, the similarity for ß-chain of HB (HBB) was 100% and 100% query coverage in species of Camelus family. The both alpha and beta chains of Hb were 84% similarity with 100% query coverage between Camelus family and Homo sapiens. The similarity between α-chain of Hb Camelus family versus Bos taurus and Equus caballus were 85% and 86% respectively. Also resemblance of ß-chain of Camelus family versus Bos taurus and Equus caballus were 83% and 82% respectively (Table 3). Therefore alpha and beta chains of hemoglobin are conserved in considered species. So it seems that, depends on common function of hemoglobin in order transferring oxygen by ferrous.


Table 3 Genome homology of α-chain (HBA) and ß-chain (HBB) of hemoglobin between mentioned species. The criterion of identity as homology with E-value and query coverage is shown for each comparison between species


2.5 Protein structure modeling
The prediction of tertiary structure for alpha and beta chains (HBA and HBB) based on homology- modelling using the ExPASy web server has shown in Figure 3. Tertiary structure of HBA was quite similar between Camelus family and N-linked glycosylation was Asn that illustrated in Figure 3. It might be caused by each mutations happened into α-chain of Hb. So through our findings, most critical functions of hemoglobin for instance oxygen transportation doesn’t been accomplished by other kinds of subunits such as gamma, delta and zeta. In fact α and ß-chains probably controlled most critical functions of hemoglobin. As showed in Figure 3 tertiary structure of HBB and HBA were all the same in Camelus family but it was different than Homo sapiens (Figure 3). As on Figure 4, the ligands of HBA and HBB in the mentioned species were heme. Role of heme is linked to oxygen, both hemoglobin subunits affected to oxygen transport. We have seen in this article that the based on homology modeling, tertiary structure of HBB in Camelus family are much likely to α-chain of the Homo sapiens (Figure 3). Despite the fact that, the based on Figure 3, HBB in Camelus family have not susceptibility position to glycosylation. As seen in Figure 3, the Asn131 and Asn132 are susceptible amino acids to N-glycosylation in HBA of domestic and wild camels respectively however all Asn in human HBA are resistance to N-glycosylation. According to similar structure between human HBA and camel HBB, we conclude that the resistivity against N-glycosylation is done by HBA in human. Whereas we demonstrated as Figure 3, HBB has less position for N-glycosylation than HBA; we conclude HBB is more involved in critical actions than HBA. As previously mentioned sequence of HBB is highly conserved in all considered species, perhaps this explains the crucial role of hemoglobin in oxygen transports.


Figure 3 The tertiary structure of predicted model for the whole sequences. 3A) HBA Camelus ferus. 3B) HBA Camelus bactrianus and dromedarius. 3C) HBA Homo sapiens. 3D) HBB Camelus ferus, Camelus bactrianus and dromedarius. 3E) HBB Homo sapiens. The predicted 3D model(s) for every domain are shown, that each chains of hemoglobin only have one domain (domain 1) in the mentioned species. As shown in Fig. 3 A and B, Asn is pottential N-glycosylated in camel but Thr is pottential O-glycosylated in Fig. 3 C, D and E. The ligand of hemoglobin (Heme) is illustrated too


2.6 Glycosylation sites
The prediction of glycation which is a non-enzymatic binding of glucose to the protein (as in the case of HbA1c) was done based on binary profile of patterns (BPP). The result of glycosylation sites for α and ß chains is shown in the Table 4. In this research, no C-linked glycosylation is found for any HBA and HBB in mentioned species. The C-linked glycosylation is comparatively rare event and in this the glycan is found attached to carbon of the first Trp residue in the consensus sequence W-X-X-W or W-X-X-C or W-X-X-F (where X is any AAs) (Krieg et al., 1998). Based on Table 4 for N-glycosylation in HBA we found a potential glycosylated site for Asn131 in Camelus bactrianus and dromedarius showed and Asn132 for Camelus ferus but Homo sapiens show no potential N-glycosylated site for both hemoglobin subunits (Table 4). N-linked glycosylation is recognized by addition of glucose to a nitrogen atom, usually the N4 of Asn that specifically recognizes a consensus sequence Asn-X-Ser/Thr, where X is any amino acid except prolin (Gavel and von Heijne, 1990). In this study we found most parts of consensus sequence which were susceptible to O-glycosylation for instance in HBA potential O-glycosylated as illustrated in Ser3-Ser4-Thr8 for domestic camels and Ser4-Thr42 for human (Table 4). The based on Table 4, alpha hemoglobin is potentially susceptible to O-glycosylation specially Camelus ferus. As showed O-glycosylation for HBB in Table 3, we found potential glycosylated only for Thr5 in Homo sapiens (Table 4). No position is susceptible to O-glycosylation in camel HBB. O-glycosylation occurs by the addition of glucose to free hydroxyl group containing AAs residues that includes Ser, Thr and to some extent, hydroxylproline and hydroxylysine (Christlet et al., 2001). Therefore we conclude that O-linked glycosylation occurs most than N-,C-linked glycosylation in HBA, and but HBB is resistant to N,O-glycosylation specially in camels however alpha hemoglobin is most susceptible to O-linked glycosylation, maybe it depends on alcoholic factor in camel and human amino acids.


Table 4 The predicted N-, O- and C-Glycosylation sites of hemoglobin (Alpha and Beta subunits) in Camelus family and human (Homo sapiens). Glycosylation type, position of glycosylation in consensus sequence and prediction of glycosylation are illustrated following


2.7 Analysis of PSI-BLAST (Position-Specific Iterated BLAST)
The result of PSI-BLAST (Position-Specific Iterated BLAST) for α and ß chains hemoglobin of domestic camels (HBA and HBB) are shown in Figure 4. We found a lot of HomoloGene between camel HB versus other species such as Pteropus alecto, Ailuropoda melanoleuca, Callithrix jacchus and etc (Figure 4). Anyway it was interesting when we found hemoglobin in domestic camel has a lot of HomoloGene versus different mammalian even Pteropus alecto or bat.


Figure 4 PSI-BLAST (Position-Specific Iterated BLAST) between α-chain (HBA) and ß-chain (HBB) Camelus ferus, dromedarius and bactrianus with different species from GenBank. We found a lot of HomoloGene from different species that each them have identity with α-chain and ß-chain hemoglobin of Camelus ferus, dromedarius and bactrianus (HBA). Maximum score, total score, query cover, E-value and ident are shown as genome homology


3 Conclusions
The findings of the current study led us to conclude that camel HBB appeared to be no susceptible to N-glycosylation than human. Based on isoelectric point, camel HBB and HBA has high mobility, and thus affects to glycosylation. We know alpha and beta subunits of hemoglobin have a lot functions such as biological process, molecular function and involvement in disease but most importantly, involved in oxygen transport from the lung to the peripheral tissues. Despite, the heme is common ligand of HBA and HBB in all species; it can link to the oxygen by ferrous. As well as, O-linked glycosylation occurs than N, C-linked glycosylation; however camel HBB is resistant to O-linked glycosylation. Although, alpha chains of hemoglobin is more susceptible to O-glycosylation. The both hemoglobin subunits are resistance to N-glycosylation. Eventually, these factors, together with other post-translational modification, might be responsible for protecting hemoglobin against glycosylation. Eventually, we believed that a particular feature of camel depends on different versions of proteins. Even, we suggest that a study on the unique features of proteins would be appropriate for identifying function and efficiency of polypeptides and subsequently production of synthetic drugs against diabetes, Parkinson, Alzheimer and others by protein engineering.
Acknowledgment
We would be grateful from Dr. Hassani-Bafrani, PhD candidate of Guilan university who has assisted us in editing English grammar of this article.
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