Galactoside-binding Proteins Research Articles

Generation of H2O2 by human neutrophils and changes of cytosolic Ca2+ and pH of rat thymocytes in response to galactoside-binding proteins (lectins or immunoglobulins).

In contrast to plant agglutinins, biological activities of animal/human lectins are not well defined yet. Testing a panel of seven mammalian carbohydrate-binding proteins we have found that the dimeric lectin from chicken liver (CL-16) was a stimulator of H2O2 release from human neutrophils as well as effector for induction of cytosolic Ca2+ and pH increase in rat thymocytes. Activity of this lectin was comparable to potent galactoside-specific plant lectins such as Viscum album L. agglutinin. The activities of the tested plant lectins depended significantly on their nominal carbohydrate specificity as well as on the source. The results indicate that endogenous lectins may be involved in the regulation of neutrophil and lymphocyte functions by elicitation of selective biosignaling reactions.

Identification of different galectins by immunoblotting after two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients

Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.

OvGalBP, a filarial antigen with homology to vertebrate galactoside-binding proteins

The increased incidence of “allergic” symptomatology and clinical complications seen in non-endemic individuals with loiasis, as compared to natives of endemic areas, is thought to reflect a heightened immune response to filarial antigens. To identify antigens involved in this hyperresponsiveness, a cDNA library constructed from adult female RNA from the related filarial parasite, Onchocerca volvulus, was screened with serum from a North American who acquired loiasis in West Africa. Sequence analysis of one of the identified clones, OvGalBP, revealed significant homology to the vertebrate S-type lectins, a family of thiol-dependent, metal-independent galactoside binding lectins, which includes an IgE-binding protein thought to be involved in IgE regulation. The 1100-bp insert of OvGalBP contains the entire protein coding region and has a 3′ poly(A) tail. The two amino acid consensus sequences (WGxExR and HFNPRF) found in all of the S-type lectins are present. Purified recombinant protein expressed as a fusion with glutathione- S-transferase (OvGalBP-GST) was recognized by sera from a majority of filaria-infected patients but not by putatively immune individuals from an endemic area or by unexposed endemic and non-endemic controls. Interestingly, OvGalBP-GST specifically bound 1gE (and not IgG) in a lactose-inhibitable manner, suggesting a potential role for this protein in the pathophysiology of human filarial infection.

Dichotomy in the laminin-binding properties of soluble and membrane-bound human galactoside-binding protein

Recent studies indicate that galactoside-binding proteins may bind the poly-N-acetyllactosamine sequences of laminin. We questioned whether human galactoside-binding protein (hL-31) binds to laminin and whether cells that express hL-31 on their surface use it as a laminin receptor to promote cellular attachment. The data show that both lectin and cells bind to immobilized laminin. The binding of soluble lectin to laminin is inhibited by lactose, while cell adhesion to it is not. The results indicate that laminin may be a ligand for soluble galactoside-binding proteins.

Sugar receptors of different types in human metastases to lung and liver.

Endogenous sugar receptors of human tumors, supposedly involved in recognitive interactions and growth regulation, were comparatively analyzed from human metastases to lung and liver by affinity chromatography and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These profiles of sugar receptors including Ca2+-dependent and Ca2+-independent specificities to alpha- and beta-galactosides, alpha-mannosyl and alpha-fucosyl moieties from salt and detergent extracts were found to be significantly different from the profile of the corresponding normal tissue. Metastatic lesions to lung from three different types of primary tumors revealed primarily tumor-associated mannan- and galactoside-binding proteins, whereas different liver metastases showed a tendency towards preferential expression of additional beta-galactoside-binding proteins and, to a reduced extent, fucose-binding proteins. The patterns of two metastatic lesions to lung and liver from a similar primary tumor, a colon carcinoma, disclose significant differences. Each resembles the pattern of other metastases to the same target organ more than it resembles the pattern of metastatic lesions to the other target organ, derived from a similar primary tumor. Further analyses of two primary liver tumors underscore the significance of changes in such a pattern upon malignant transformation.

Carbohydrate-binding proteins of tumor lines with different growth properties

A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weight of 42 kDa. The alpha-glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5-8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5-8#1 and 5-20#20, whereas the metastatic variant 5-20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.

Ricin-like plant toxins are evolutionarily related to single-chain ribosome-inhibiting proteins from Phytolacca.

A comparison has been made of the amino-terminal sequences of a number of ribosome-inhibiting proteins (RIPs) and cytotoxins. These include the monomeric enzymes PAP, PAP-S, PAP-II, and dodecandrin and the enzymatic A chains from the heterodimeric toxins ricin and modeccin. We show that these proteins have all evolved from a single ancestor. A statistical analysis is used to show the likely evolutionary relationship among the proteins. A similar analysis was performed on the amino-terminal sequences of ricin, Ricinus agglutinin, and modeccin B chains. These are galactoside-binding proteins associated with the A-chain enzymes. From the two comparisons we propose a scheme for the development of two major classes of proteins. The RIP and sugar-binding genes probably evolved independently. In some plant lines the genes never fused, although the RIP gene replicated and developed into several proteins expressed at various stages of plant maturation. In another line the RIP gene fused with a sugar binding (B-chain) gene to form the class of heterodimeric toxins. In some species this fused gene appears to have multiplied, one or more of the toxin genes mutating to code for a self-dimerizing agglutinin molecule.