title: Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2(SNAT2) by Chemical Modification.
abstract: Sodium-coupled neutral amino acid transporter 2 (SNAT2) belongs to solute carrier 38 (SLC38) family of transporters, which is ubiquitously expressed in mammalian tissues and mediates transport of small, neutral amino acids, exemplified by alanine(Ala, A). Yet structural data on SNAT2, including the relevance of intrinsic cysteine residues on structure and function, is scarce, in spite of its essential roles in many tissues. To better define the potential of intrinsic cysteines to form disulfide bonds in SNAT2, mutagenesis experiments and thiol-specific chemical modifications by N-ethylmaleimide (NEM) and methoxy-polyethylene glycol maleimide (mPEG-Mal, MW 5000) were performed, with or without the reducing regent dithiothreitol (DTT) treatment. Seven single mutant transporters with various cysteine (Cys, C) to alanine (Ala, A) substitutions, and a C245,279A double mutant were introduced to SNAT2 with a hemagglutinin (HA) tag at the C-terminus. The results showed that the cells expressing C245A or C279A were labeled by one equivalent of mPEG-Mal in the presence of DTT, while wild-type or all the other single Cys to Ala mutants were modified by two equivalents of mPEG-Mal. Furthermore, the molecular weight of C245,279A was not changed in the presence or absence of DTT treatment. The results suggest a disulfide bond between Cys245 and Cys279 in SNAT2 which has no effect on cell surface trafficking, as well as transporter function. The proposed disulfide bond may be important to delineate proximity in the extracellular domain of SNAT2 and related proteins.
authors.affiliation: College of Life Science and Biopharmacy, Shenyang Pharmaceutical University, Shenyang 110015, People's Republic of China.
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.affiliation: College of Life Science and Biopharmacy, Shenyang Pharmaceutical University, Shenyang 110015, People's Republic of China.
authors.forename: Jiahong
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Life Science and Biopharmacy, Shenyang Pharmaceutical University, Shenyang 110015, People's Republic of China.
authors.forename: Ruiping
authors.initials: R
authors.lastname: Cai
authors.affiliation: College of Life Science and Biopharmacy, Shenyang Pharmaceutical University, Shenyang 110015, People's Republic of China.
authors.forename: Yanmeng
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, People's Republic of China.
authors.forename: Zhanyun
authors.initials: Z
authors.lastname: Guo
authors.affiliation: Departments of Chemistry and Biological Sciences, Binghamton University, Binghamton, New York, 13902, United States of America.
authors.forename: Christof
authors.initials: C
authors.lastname: Grewer
authors.affiliation: College of Life Science and Biopharmacy, Shenyang Pharmaceutical University, Shenyang 110015, People's Republic of China.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000409:Alanine / Q000737:chemistry; D026924:Amino Acid Transport System A / Q000737:chemistry*; D026905:Amino Acid Transport Systems / Q000737:chemistry*; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D017136:Ion Transport; D016296:Mutagenesis; D009154:Mutation; D011092:Polyethylene Glycols / Q000737:chemistry; D051381:Rats; D012964:Sodium / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D026924:Amino Acid Transport System A; D026905:Amino Acid Transport Systems; D004220:Disulfides; C498797:SLC38A2 protein, human; C412568:Slc38a2 protein, rat; D013438:Sulfhydryl Compounds; D011092:Polyethylene Glycols; D012964:Sodium; D003545:Cysteine; D005033:Ethylmaleimide; D000409:Alanine; D004229:Dithiothreitol