Purification of Recombinant Galectins from Different Species Using Distinct Affinity Chromatography Methods.

Galectins are lectins having the capacity to recognize ß-galactose-containing glycan structures and are widely distributed among various taxa. However, the exact physiological and biochemical functions mediated by galectins that necessitate their wide occurrence among diverse species have not yet been delineated in a precise manner. Purification of recombinant galectins in active form is a fundamental requirement to elucidate their biological function. In this chapter, we are describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinerea galectin 2 (CGL2), nickel-chromatography for histidine-tagged human galectin-7, and glutathione-Sepharose chromatography for Glutathione S-transferase-tagged (GST-tagged) human galectin-7. Step-by-step instructions are provided for obtaining the above-mentioned recombinant galectins that retain carbohydrate-binding activity and are suitable for conducting biochemical experiments.

Marginal zone B cells mediate a CD4 T-cell-dependent extrafollicular antibody response following RBC transfusion in mice.

Red blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme [HEL] and ovalbumin [OVA] fused with the human RBC antigen Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they colocalize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited immunoglobulin M (IgM) and IgG anti-HOD antibody formation, whereas CD4 T-cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild-type or MZ B-cell-deficient recipients, suggesting that IgG formation is not dependent on MZ B-cell-mediated CD4 T-cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response, and no increase in antigen-specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest that MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

Generation and Use of Recombinant Galectins.

Galectins are soluble carbohydrate binding proteins that can bind ß-galactose-containing glycoconjugates by means of a conserved carbohydrate recognition domain (CRD). In mammalian systems, galectins have been shown to mediate very important roles in innate and adaptive immunity as well as facilitating host-pathogen relationships. Many of these studies have relied on purified recombinant galectins to uncover key features of galectin biology. A major limitation to this approach is that certain recombinant galectins purified using standard protocols are easily susceptible to loss of glycan-binding activity. As a result, biochemical studies that employ recombinant galectins can be misleading if the overall activity of a galectin remains unknown in a given assay condition. This article examines fundamental considerations when purifying galectins by lactosyl-sepharose and nickel-NTA affinity chromatography using human galectin-4N and -7 as examples, respectively. As other approaches are also commonly applied to galectin purification, we also discuss alternative strategies to galectin purification, using human galectin-1 and -9 as examples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Purification of galectins using lactosyl-sepharose affinity chromatography Basic Protocol 2: Purification of human galectin-7 using a nickel-NTA affinity chromatography column Alternate Protocol 1: Iodoacetamide alkylation of free sulfhydryls on galectin-1 Alternate Protocol 2: Purification of human galectin-9 using lactosyl-sepharose column chromatography.