title: Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin.
abstract: Sugar binding studies, inactivation, unfolding, and refolding of native Jacalin (nJacalin) from Artocarpus integrifolia and recombinant single-chain Jacalin (rJacalin) expressed in Escherichia coli were studied by intrinsic fluorescence and thermal and chemical denaturation approaches. Interestingly, rJacalin does not undergo any proteolytic processing in an E. coli environment. It has 100fold less affinity for methyl-alpha-galactose (Ka: 2.48 x 10(2)) in comparison to nJacalin (Ka: 1.58 x 10(4)), and it also binds Thomsen-Friedenreich (TF) disaccharide (Galbeta1-3GalNAc) with less affinity. Overall sugar binding characteristics of rJacalin are qualitatively similar to that of nJacalin (Gal<MealphaGal<MealphaTFdisaccharide). Circular dichroism studies at near- and far-UV, thermal, and chemical denaturation studies reveal that the rJacalin behaves like nJacalin. Guanidine hydrochloride-induced denaturation, followed by renaturation, yielded total recovery of sugar binding activity of rJacalin in comparison to partial recovery for nJacalin. This signifies the minor changes in the refolding pathways between native and recombinant lectins. The stability of rJacalin is dramatically reduced in the extreme pH range unlike nJacalin. Both lectins do not bind 1-anilino-8-naphthalene sulfonic acid (ANS) in the pH range of 5 to 12 but they do in the pH range of 1-3. Solute quenching studies of the lectin using acrylamide, KI, and CsCl indicated that the tryptophan residues have full accessibility to the neutral quencher and poor accessibility to ionic quenchers. In summary, biophysical and biochemical studies on the native versus recombinant Jacalin suggest that post-translational modification, i.e., the processing of Jacalin into two chains is probably not a prerequisite for sugar binding but may be required for higher affinity.
authors.affiliation: National Centre for Cell Science, Pune, Maharashtra, India.
authors.forename: Anagh A
authors.initials: AA
authors.lastname: Sahasrabuddhe
authors.forename: Sushama M
authors.initials: SM
authors.lastname: Gaikwad
authors.forename: M V
authors.initials: MV
authors.lastname: Krishnasastry
authors.forename: M Islam
authors.initials: MI
authors.lastname: Khan
mesh_terms: D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002942:Circular Dichroism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008756:Methylgalactosides / Q000378:metabolism; D008969:Molecular Sequence Data; D037121:Plant Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D008756:Methylgalactosides; D037121:Plant Lectins; D011994:Recombinant Proteins; C043696:jacalin