Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages.
Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.
Aruna
Battu
B
Molecular Biology Unit, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania PO, Hyderabad 500007, India.
Ghosh
Sudip
S
Singh
Anil K
AK
Mande
Shekhar C
SC
Srinivas
V
V
Chauhan
Radha
R
Ehtesham
Nasreen Z
NZ
D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D006729:Hormones, Ectopic / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008623:Mercaptoethanol / Q000737:chemistry; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D052243:Resistin; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D004220:Disulfides; D006729:Hormones, Ectopic; D036341:Intercellular Signaling Peptides and Proteins; C499624:RETN protein, human; C486574:RETNLB protein, human; D011994:Recombinant Proteins; D052243:Resistin; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D014508:Urea