Extracellular Gal-3 and oligodendroglial differentiation

THOMAS L.1, MARZIALI L.N. 1, RABINOVICH G.A.2, PASQUINI J.M.1, PASQUINI L.A.1

Congreso; SAIC; 2016

Extracellular Gal-3 and oligodendroglial differentiationThomas L.1, Marziali L.N. 1,Rabinovich G.A.2, Pasquini J.M.1, Pasquini L.A.1 1Departmentof Biological Chemistry, School of Pharmacy and Biochemistry, Institute of Chemistryand Biological Physicochemistry (IQUIFIB), School of Pharmacy and Biochemistry,University of Buenos Aires and National Research Council (CONICET), Argentina.2Laboratoryof Immunopathology, Institute of Biology and Experimental Medicine (IBYME; CONICET),C1428 Buenos Aires, Argentina and Department of Biological Chemistry, School ofExact and Natural Sciences, University of Buenos Aires, C1428 Buenos Aires,Argentina Gal-3, a chimericprotein structurally composed of unusual tandem repeats of proline and short glycine-richsegments fused onto a carbohydrate recognition domain, possesses multifacetedroles in physiological processes including the regulation of innate andadaptive immune responses (Rabinovich et al, 2012).  Previous results have demonstrated that recombinantGal-3 (rGal-3) treatment accelerates oligodendrocyte (OLG) differentiation in adose- and carbohydrate-dependent manner, which is in accordance with the N-glycosylationprofile observed in immature versus differentiated OLG (Pasquini et al, 2011). Theaim of our work is to elucidate the mechanism involved in Gal-3-induced OLG differentiationby means of rGal-3 obtained and purified optimizing the standard protocol forrecombinant protein production. rGal-3 was obtained from the Escherichia coli BL21 star/pET28b+-Gal-3 system. Inducerconcentration and period of induction were improved to increase the overallyield. rGal-3 release from bacteria was optimized through changes in ultrasonicationtime and acoustic power. rGal-3 was purified by affinity chromatography withagarose-lactose and bacterial LPS was removed by affinity chromatography with polymyxinb. Protein biological activity was controlled in every step by hemagglutinationand finally by the evaluation of the dose-dependent viability of treated OLG. Theevaluation of migration in vitro showedcells treated with Gal-3 to migrate significantly less than control cells. Also, ex vivo mice brain slices were used inorder to study the effect of Gal-3 on myelination, dysmyelination induced byLPC and remyelination after LPC, both in wild type and LGALS-3-/-. Results indicate that Gal-3 is a key factorto achieve correct myelination, and that the rGal-3 obtained is biologicallyactive to induce correct oligodendroglial maturation.   .