The promiscuous binding pocket of SLC35A1 ensures redundant transport of CDP-ribitol to the Golgi

Abstract

The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency. The glycoprotein α-dystroglycan helps to link the intracellular cytoskeleton to the extracellular matrix. A unique glycan structure attached to this protein is required for its interaction with extracellular matrix proteins such as laminin. Up to now, this is the only mammalian glycan known to contain ribitol phosphate groups. Enzymes in the Golgi apparatus use CDP-ribitol to incorporate ribitol phosphate into the glycan chain of α-dystroglycan. Since CDP-ribitol is synthesized in the cytoplasm, we hypothesized that an unknown transporter must be required for its import into the Golgi apparatus. We discovered that CDP-ribitol transport relies on the CMP-sialic acid transporter SLC35A1 and the transporter SLC35A4 in a redundant manner. These two transporters are closely related, but bulky residues in the predicted binding pocket of SLC35A4 limit its size. We hypothesized that the large binding pocket SLC35A1 might accommodate the bulky CMP-sialic acid and the smaller CDP-ribitol, whereas SLC35A4 might only accept CDP-ribitol. To test this, we expressed SLC35A1 with mutations in its binding pocket in SLC35A1 KO cell lines. When we restricted the binding site of SLC35A1 by introducing the bulky residues present in SLC35A4, the mutant transporter was unable to support sialylation of proteins in cells but still supported ribitol phosphorylation. This demonstrates that the size of the binding pocket determines the substrate specificity of SLC35A1, allowing a variety of cytosine nucleotide conjugates to be transported. The redundancy with SLC35A4 also explains why patients with SLC35A1 mutations do not show symptoms of α-dystroglycan deficiency. The dystrophin-associated glycoprotein 1 (DAG1) gene gives rise to a single protein that is subsequently cleaved into two fragments that remain attached to each other. The C-terminal part, β-dystroglycan, is a transmembrane protein anchored to the actin cytoskeleton through interaction with the dystrophin protein (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1158) Google Scholar). The N-terminal extracellular part, α-dystroglycan, binds to extracellular matrix components, like laminin, agrin, perlecan, or neurexin (1Ibraghimov-Beskrovnaya O. Ervasti J.M. Leveille C.J. Slaughter C.A. Sernett S.W. Campbell K.P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.Nature. 1992; 355: 696-702Crossref PubMed Scopus (1158) Google Scholar). This interaction depends on the assembly of a complex glycan on α-dystroglycan. Defects in several enzymes required for the biogenesis of this glycan lead to a group of congenital syndromes characterized by muscle, brain, and eye symptoms. These syndromes have been coined dystroglycanopathies and present a vast clinical spectrum ranging from mild muscular weakness with late onset to severe congenital muscular dystrophy with brain and eye involvement (muscle–eye–brain disease, Walker–Warburg syndrome, and Fukuyama congenital muscle dystrophy) (2Endo T. Glycobiology of alpha-dystroglycan and muscular dystrophy.J. Biochem. 2015; 157: 1-12Crossref PubMed Scopus (88) Google Scholar, 3Live D. Wells L. Boons G.J. Dissecting the molecular basis of the role of the O-mannosylation pathway in disease: Alpha-dystroglycan and forms of muscular dystrophy.Chembiochem. 2013; 14: 2392-2402Crossref PubMed Scopus (19) Google Scholar, 4Michele D.E. Barresi R. Kanagawa M. Saito F. Cohn R.D. Satz J.S. Dollar J. Nishino I. Kelley R.I. Somer H. Straub V. Mathews K.D. Moore S.A. Campbell K.P. Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies.Nature. 2002; 418: 417-422Crossref PubMed Scopus (677) Google Scholar, 5Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (109) Google Scholar). The investigation of genes mutated in affected patients has led to the discovery of many steps in this process and a better understanding of α-dystroglycan glycosylation as well as more generally of protein O-mannosylation. The α-dystroglycan protein contains three domains: the C-terminal domain, which binds to the extracellular domain of β-dystroglycan, the mucin domain characterized by several mucin-type-O-glycosylation sites as well as a key O-mannosyl glycan important for ligand binding (referred to later as core M3), and the N-terminal domain that is required for complete glycosylation and is subsequently shed by proteolytic cleavage (5Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (109) Google Scholar). The ligand-binding epitope of α-dystroglycan is assembled on the core M3 glycan, which has been extensively studied and consists of an O-linked phosphoryl(C6)-mannose extended with a GlcNAc and a GalNAc. It is formed by sequential action of protein O-mannosyltransferases 1 and 2 (5Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (109) Google Scholar), protein O-linked mannose β-1,2-N-acetylglucosaminyltransferase (6Ogawa M. Nakamura N. Nakayama H. Kanagawa M. T. T. alpha-dystroglycan in the to with 2013; PubMed Scopus Google Scholar), 2 T. D.E. L. C.A. L. D. in congenital muscular dystrophy and of J. 2013; PubMed Scopus Google Scholar), and T. T. D. T. F. H. L. Campbell K.P. is a required for dystroglycan 2013; PubMed Scopus Google Scholar). the Golgi this glycan is extended by and protein with a of ribitol phosphate I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google Scholar, M. M. H. M. H. T. T. of a with and its in muscular 14: PubMed Scopus Google Scholar, T. D. H. Moore S.A. Campbell K.P. glycan on alpha-dystroglycan contains a for PubMed Scopus Google Scholar). This structure as a to which the is with of a of and acid that are by the enzymes transmembrane protein H. Kanagawa M. M. H. M. T. T. The muscular dystrophy gene a ribitol required for the glycosylation of PubMed Scopus Google and 1 Boons G.J. Wells L. is the for the glycosylation of Scopus Google Scholar, T. D. D. L. Campbell K.P. The is required for of alpha-dystroglycan Scopus Google for the and subsequently by the proteins or and 1 or T. L. Campbell K.P. and of PubMed Scopus Google Scholar). and in the Golgi apparatus use CDP-ribitol, which is synthesized by the protein in the I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google Scholar, M. M. H. M. H. T. T. of a with and its in muscular 14: PubMed Scopus Google Scholar, T. D. H. Moore S.A. Campbell K.P. glycan on alpha-dystroglycan contains a for PubMed Scopus Google Scholar). the transporter required for the of CDP-ribitol into the Golgi and is the of the are by proteins known as nucleotide transporters to the gene These proteins transport nucleotide into the or the Golgi apparatus the C.J. T. J. and of nucleotide J. PubMed Scopus Google Scholar). have the that are required for this transport basis for mammalian nucleotide PubMed Scopus Google Scholar). the substrate specificity of several transporters is still unknown T. C.J. T. J. transporter structure and J. PubMed Scopus Google Scholar). we are only to transporters basis for mammalian nucleotide PubMed Scopus Google Scholar, basis of nucleotide transport the Golgi PubMed Scopus Google Scholar). two that the Golgi apparatus CMP-sialic acid transporter SLC35A1 be in CDP-ribitol The of from a to required for the of which is on the interaction with α-dystroglycan. the cell the that of SLC35A1, the CMP-sialic acid glycosylation of α-dystroglycan and M. M. H. H. the of dystroglycanopathies for 2013; PubMed Scopus Google Scholar). SLC35A1 mutations in patients unable to the α-dystroglycan glycosylation in SLC35A1 KO cell M. J. T. G.J. H. mutations in CMP-sialic acid transporter SLC35A1 in alpha-dystroglycan from 2015; PubMed Scopus Google Scholar). sialylation was by a acid α-dystroglycan still to its ligand laminin, that sialylation is not required for α-dystroglycan This that the of α-dystroglycan glycosylation in SLC35A1 KO cells was not of a of CMP-sialic acid in the of the Golgi apparatus M. J. T. G.J. H. mutations in CMP-sialic acid transporter SLC35A1 in alpha-dystroglycan from 2015; PubMed Scopus Google Scholar). we that SLC35A1 and its SLC35A4 a redundant role in the glycosylation of α-dystroglycan. a of we that the large binding site of SLC35A1 the transport of CMP-sialic acid and CDP-ribitol. why patients with SLC35A1 do not show clinical symptoms a To the by M. M. H. H. the of dystroglycanopathies for 2013; PubMed Scopus Google Scholar), we SLC35A1 in cells and α-dystroglycan glycosylation by the the α-dystroglycan glycan as well as protein sialylation by and binding to acid residues of SLC35A1 led to a in and in a in protein which was of and SLC35A1 also led to a in α-dystroglycan glycosylation as by the which was also by of and To the of α-dystroglycan in SLC35A1 KO we a The a for was in SLC35A1 KO the molecular of α-dystroglycan was of a of sialylation and the of the We also that the molecular of the was with the known sialylation of this of SLC35A1 in KO cell the in binding and and the of with a complete of sialylation of the known role of SLC35A1 in CMP-sialic acid the and molecular in as well as the in with the and but not in This that the of the ribitol glycan was only the of SLC35A1 whereas to be required to the of the be sialylation was required for the of a key in the biogenesis of the has been that of sialylation by a acid not the biogenesis of the glycan M. J. T. G.J. H. mutations in CMP-sialic acid transporter SLC35A1 in alpha-dystroglycan from 2015; PubMed Scopus Google Scholar). a more is that SLC35A1 not only CMP-sialic acid but also CDP-ribitol, with a SLC35A1 KO cells still in the and in with the which the α-dystroglycan glycan and This that SLC35A1 might be the transporter for CDP-ribitol in transporters might To the of ribitol into α-dystroglycan, we which we to large of α-dystroglycan in the When we SLC35A1 in we in and a in sialylation A and We α-dystroglycan from the and ribitol by as I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google Scholar). To of ribitol into α-dystroglycan was SLC35A1 was in cells SLC35A1 was required for the of the glycan of α-dystroglycan in cells but not required for the of ribitol into α-dystroglycan in This led to that two transporters might a redundant role in CDP-ribitol transport in that transporters to the we an of of the in and This that SLC35A4, a with unknown D. M. into the nucleotide transporter PubMed Scopus Google Scholar), was expressed in cells in cells We also that a CDP-ribitol transporter be expressed in the brain and muscle, α-dystroglycan has important on gene from the M. S.A. J. C.A. novel to The 2015; PubMed Scopus Google Scholar), SLC35A1, and SLC35A4 show in muscle and brain in the brain, and We hypothesized that SLC35A1 and SLC35A4 might a redundant role in CDP-ribitol transport in To test this we to SLC35A4 in cells as well as in the SLC35A1 KO We a α-dystroglycan from the and ribitol This that of SLC35A4 not ribitol of SLC35A1 and SLC35A4 ribitol in the glycan of α-dystroglycan This is of protein in the KO cells that proteins a redundant in ribitol into α-dystroglycan. To the of redundancy of SLC35A1 and SLC35A4, we to we α-dystroglycan and We SLC35A1 KO cells with of of SLC35A4 not protein sialylation as by and A and we that α-dystroglycan by the and was SLC35A4 to we that of SLC35A1 binding to and the molecular of α-dystroglycan and SLC35A4 the in to the in only the in molecular and by SLC35A1 of SLC35A4 not the of binding A and in SLC35A1 KO that SLC35A4 not to CMP-sialic acid This is also supported by the that with led to a molecular and in and KO cell whereas was SLC35A1 KO cell we that transporters are redundant in to the of the glycan, but that only SLC35A1 transport CMP-sialic acid allowing we CDP-ribitol and CMP-sialic acid in the SLC35A1 and SLC35A4 single and KO cells by of SLC35A1 led to in CMP-sialic acid with the that this nucleotide is not of the of its transporter CDP-ribitol or SLC35A1 SLC35A4 was This is with that α-dystroglycan glycosylation in cell is not of a in CDP-ribitol but cells expressed SLC35A1, whereas cells on a redundant of SLC35A1 and SLC35A4 to the To test a redundancy might in we also the of the of two proteins in a cell that we proteins and of α-dystroglycan in and in a of more on the of SLC35A1 or SLC35A4 on the of α-dystroglycan 2 and SLC35A1 and SLC35A4 led to a of that SLC35A4 and SLC35A1 in a redundant in the assembly of the glycan of α-dystroglycan. SLC35A4 is not to the of CMP-sialic acid transport in SLC35A1 KO that SLC35A4 transport CMP-sialic of the structure of the CMP-sialic acid transporter SLC35A1 basis for mammalian nucleotide PubMed Scopus Google Scholar), which a for to the molecular of substrate in is SLC35A4 and SLC35A1 the residues in SLC35A1 that with the group of are in SLC35A4, and and A and We that two residues are in nucleotide are and in the protein of the and transporter and the transporter basis for mammalian nucleotide PubMed Scopus Google Scholar). The that residues are CMP-sialic acid transporter and SLC35A4 that SLC35A4 be to transport and CDP-ribitol, of sites in SLC35A1 and the structure of in complex with is and the of SLC35A4 is with CDP-ribitol in the or of 1 and which are in of the are for a of the structure of in complex with in the of the structure as in but with CDP-ribitol a of is in the of SLC35A4 with CDP-ribitol in the are for 1 and of for key residues are and interactions are by as a in a that is to that in a is for the structure in complex with and in a that is to that in a is for the of SLC35A4 with CDP-ribitol in the or with into the to show the that the and several a the through the only that are of the substrate are for CMP-sialic CMP-sialic acid is also SLC35A1 and SLC35A4 in the SLC35A1 with the of and CMP-sialic acid and SLC35A1 with the phosphate and the of the acid of CMP-sialic acid through several residues and A and These residues are in of the and are also important for with the and of the of transporter and transporter basis for mammalian nucleotide PubMed Scopus Google Scholar). of residues in SLC35A4 the that this protein also be to and transport the SLC35A4 not with SLC35A1 in the the binding site for the acid of CMP-sialic acid and This in SLC35A1 is which is to accommodate the bulky acid and We that of the substrate specificity in CMP-sialic acid from interactions the protein and the nucleotide of the are not many interactions the protein and the acid of CMP-sialic acid and also with and whereas acid basis for mammalian nucleotide PubMed Scopus Google Scholar). this of specificity to a might be not only the but also R. of the acid Biochem. PubMed Scopus Google Scholar). It also that SLC35A1 be to transport the is smaller a acid CDP-ribitol, CDP-ribitol into SLC35A1 that is to accommodate a in the site of SLC35A4 that its is smaller the in SLC35A1 This is on of the of residues with to with and in SLC35A1 are and in SLC35A4 and CDP-ribitol into the of SLC35A4 that CDP-ribitol in two which we and and These binding to be by the that the the ribitol is such that is to the of the with and and the extended the ribitol to the of the with and A smaller be important for the ribitol of CDP-ribitol, which is smaller a of the of SLC35A4 with the CMP-sialic SLC35A1 structure that and with the acid that SLC35A4 not be of CMP-sialic To test the that the size of the pocket of SLC35A1 explains its to transport CMP-sialic acid and CDP-ribitol, we a of in SLC35A1 that limit the size of its pocket we expressed SLC35A1 or SLC35A4 in SLC35A1 KO To that the to transport CMP-sialic we which unable to CMP-sialic acid in SLC35A1 KO cells we a to which transporters still CDP-ribitol This that SLC35A1 still the of the glycan to an that was to the SLC35A1 was that still CDP-ribitol three unable to CMP-sialic acid in SLC35A1 KO cells that the to transport CMP-sialic a more we on mutations and as well as that like the protein we sialylation with or to SLC35A4, the and unable to the or the in SLC35A1 KO whereas the mutant and SLC35A1 to do This that mutations and transport of CMP-sialic acid by the size of the binding an be that mutations transport To test this we glycosylation of α-dystroglycan the and a that α-dystroglycan glycosylation depends on the transport of CDP-ribitol into the Golgi we the of SLC35A1 KO we that of and to a as This that still to transport CDP-ribitol unable to transport CMP-sialic the three that to the sialylation and only the of the size of α-dystroglycan in SLC35A1 KO cells that of the size in SLC35A1 KO cell is to the of sialylation the that mutations and the binding site and limit the substrate spectrum of SLC35A1 to CDP-ribitol. The sequential of two ribitol phosphate residues is required to the glycan on α-dystroglycan. the cytoplasm, the protein CDP-ribitol, which is by and in the Golgi apparatus. was still unknown CDP-ribitol was into the Golgi apparatus. this we that SLC35A1, the Golgi CMP-sialic acid and SLC35A4, a transporter of unknown are in the transport of CDP-ribitol into the Golgi apparatus. M. M. H. H. the of dystroglycanopathies for 2013; PubMed Scopus Google Scholar, M. J. T. G.J. H. mutations in CMP-sialic acid transporter SLC35A1 in alpha-dystroglycan from 2015; PubMed Scopus Google that of the CMP-sialic acid transporter SLC35A1 in cells led to a α-dystroglycan glycosylation of its role in sialylation M. J. T. G.J. H. mutations in CMP-sialic acid transporter SLC35A1 in alpha-dystroglycan from 2015; PubMed Scopus Google Scholar). that CDP-ribitol to a CMP-sialic was to that SLC35A1 also as a CDP-ribitol in SLC35A1 in lead to and T. F. R. H. D. M. in the gene for the acid transporter SLC35A1 is required for but not PubMed Scopus Google Scholar, M. J. M. of for by novel mutations in the CMP-sialic acid J. PubMed Scopus Google Scholar, M. M. R. and to acid 2013; PubMed Scopus Google Scholar). of SLC35A1 in led to H. J. S.A. J. L. to and in the Google Scholar). from affected patients show a in sialylation and CMP-sialic acid into the Golgi apparatus M. J. M. of for by novel mutations in the CMP-sialic acid J. PubMed Scopus Google Scholar). was molecular or clinical that patients from a in α-dystroglycan of SLC35A1 the of α-dystroglycan glycosylation in SLC35A1 KO that SLC35A1 might have a role in CDP-ribitol but the that in many cell this transport be by the present we this by that SLC35A1 and SLC35A4 a redundant role in allowing ribitol phosphate into the glycan of α-dystroglycan. are with the of transporters to CDP-ribitol which might for the of the glycan in SLC35A1 KO cells and and a why patients with mutations in SLC35A4 have been Up to now, is known the of on its with of the has been to be a transporter T. C.J. T. J. transporter structure and J. PubMed Scopus Google Scholar). of SLC35A4 not lead to in protein glycosylation D. M. into the nucleotide transporter PubMed Scopus Google Scholar, F. M. T. M. and nucleotide transporters in Golgi in PubMed Scopus Google Scholar). an to the of SLC35A4, D. M. into the nucleotide transporter PubMed Scopus Google to molecular and a of proteins that SLC35A4 is not with the of of intracellular that the of SLC35A4 might show a in the SLC35A1 structure and predicted These be as that SLC35A4 not be a might in to we show that SLC35A4 the ribitol into α-dystroglycan in SLC35A1 KO have been the from on the of an epitope not protein but in interactions or protein is that SLC35A4 protein with the of the SLC35A4 structure on the SLC35A1 structure that SLC35A4 to CDP-ribitol that SLC35A1 and SLC35A4 a redundant role in ribitol to the why two transporters for CDP-ribitol in the Golgi apparatus. the structure of SLC35A1 that substrate by interactions with the nucleotide of the nucleotide basis for mammalian nucleotide PubMed Scopus Google Scholar). that the the nucleotide this the of acid is and more which is is not that CDP-ribitol in the binding pocket of SLC35A1, this transporter might be to CMP-sialic the large pocket of SLC35A1 has to accommodate CMP-sialic acid have in as might also to be R. of the acid Biochem. PubMed Scopus Google Scholar). This led to that the SLC35A1 and SLC35A4 might be the size of the binding we into the sites residues the that are present in with such mutations unable to the sialylation in SLC35A1 KO cells but still α-dystroglycan glycosylation ribitol of the nucleotide to be of key for basis for mammalian nucleotide PubMed Scopus Google Scholar, basis for substrate specificity and of nucleotide transporters in the PubMed Scopus Google Scholar). the structure of the to be transporters transport a variety of conjugates I. H. T. D. F. M. Golgi transporter of PubMed Scopus Google Scholar, T. C.J. M. and of a transporter from 2015; PubMed Scopus Google Scholar). that the size of the binding pocket might be a in of bulky might be a but SLC35A1 that might well be We a from and the by a C-terminal and sites the and The was with the and and into the and sites of the in the The is a on the is by a that of The was by with as a a C-terminal and sites the and The was the and into the and to the for mutant and by assembly this, two fragments of the by the to in mammalian the assembly was with the with the enzymes and The to genes into the site of the and of J. V. F. the 2013; PubMed Scopus Google Scholar). The for each gene is in with the in SLC35A1 and SLC35A4 into sites of a which is to the by T. V. M. M. J.S. N. J. for 2013; PubMed Scopus Google Scholar). by of two in the by the and in to with and subsequently with of cell in and with 1 of in for in with for with for or with for cells as and for with for the cells with or with of for the cells with cells as in and a cells from in and cells a from of and in 2 and To KO we cells with and 1 of in cells several in and with 2 for later and into was from the the predicted cleavage site was and the by I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google Scholar). for or by cells with and from and as well as a the phosphate as M. mammalian of PubMed Scopus Google Scholar). cells in the of cells for 2 with or cells 2 for for and or for for The of the glycan of dystroglycan was by and I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google Scholar). we from cells that been to an N-terminal of α-dystroglycan an of and a I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google Scholar). proteins with acid and with of a was by in of and for 1 the and with for I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google of was in as the a of 1 The was for and to The and and in and by by of and fragments with the for The the of and subsequently to the are to the ribitol in as I. I. J. M. J. D. N. J.M. I. CDP-ribitol by and to ribitol phosphate PubMed Scopus Google Scholar). proteins in and and by protein the acid protein of protein for with to three with 1 of for in the of and M. F. I. R. F. for the of proteins of and molecular PubMed Scopus Google to proteins on to for V. with was the a of for with 1 and 1 for 1 and with in three for with and for 2 with in three with for 1 with in a I. A for and PubMed Scopus Google three more in to the the of the the molecular was with a of the and be in the was as M. N. 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