Multiple soluble beta-galactoside-binding lectins from human lung

Soluble extracts of human lung contain three major beta-galactoside-binding proteins with apparent subunit molecular weights of 14,000 (HL-14), 22,000 (HL-22), and 29,000 (HL-29). HL-14 and HL-29 were abundant in all the specimens that we tested whereas HL-22 was abundant in some and very scarce in others. HL-14 could be resolved into at least six acidic forms by isoelectric focusing and HL-29 into at least five acidic forms by this procedure. In contrast, HL-22 is a basic protein. Other beta-galactoside-binding proteins with subunit molecular weights ranging from about 16,000 to 27,000 were also detected in lung extracts, but the possibility that they are degradation products cannot be excluded. HL-14 is very similar to a rat lung lectin (RL-14.5) in carbohydrate binding specificity and amino acid composition and reacts strongly with an antiserum raised against the rat lectin. HL-29 is similar to the rat lectin RL-29 in the same respects, but its carbohydrate binding specificity is somewhat different. Of the known rat lectins, HL-22 resembles RL-18 most closely in carbohydrate binding specificity, but it is significantly different in other properties and does not react with an antiserum raised against the rat lectin.     

Identification and synthesis of a novel 15 kDa beta-galactoside-binding lectin in human leukocytes.

Knowledge of the identity, synthesis and secretion of beta-galactoside-binding lectins by leukocytes is of importance because lactosaminoglycans present at the leukocyte cell surface may be physiologically significant lectin receptors that could mediate autocrine or paracrine functions and/or cell adhesion. This paper presents data that show that a previously identified 15.5-16.5 kDa lactose-binding protein synthesized in vitro by human peripheral leukocytes is actually comprised of three different polypeptides. One of these is related to a novel 15 kDa lectin isolated from human spleen and which is synthesized by B lymphoblastoid cells. Spleen contains at least six lactose-binding polypeptides for which the carbohydrate-binding activity is independent of the presence of divalent cations and mercaptoethanol. The splenic 15 kDa polypeptide does not appear to be immunologically related to previously characterized beta-galactoside-binding lectins. It is separable from galaptin, another galactoside-binding lectin (subunit mol. wt 14.5 kDa) by chromatography on DEAE-Sephacel. Western blot analyses and immunoprecipitation/fluorography experiments with metabolically labelled cells showed the presence of the 15 kDa lectin in peripheral leukocytes and in Epstein-Barr virus-immortalized B lymphoblastoid cells. The 15 kDa lectin yielded polypeptide fragments of approximately 6.2 and approximately 8.6 kDa after cyanogen bromide (CNBr) degradation. These fragments were partially sequenced and 12 residues/fragment were identified. A similarity search of the SWISS PROT protein data base did not reveal a relationship of the 15 kDa polypeptide to known lectins, including galaptin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of endogenous sugar-binding proteins (lectins) in human placenta by histochemical localization and biochemical characterization

Human placentas of different stages of development were histochemically analyzed for expression of endogenous sugar-binding proteins using a panel of biotin-conjugated, chemically glycosylated probes with specificity for beta-galactosides, alpha-galactosides, alpha-mannosides, alpha-fucosides and alpha-glucosides. Temporal differences in the expression of sugar-binding proteins and different patterns of staining of the component cell types of human placenta were discerned, especially pronounced for alpha-fucoside-specific binding in the trophoblast and alpha-glucoside-specific binding in fetal and maternal macrophages. Fractionation of salt and detergent extracts from human placentas by affinity chromatography on columns with immobilized carbohydrates or glycoproteins substantiated the histochemically detectable temporal changes on the basis of alterations in the pattern of individual sugar-binding proteins, as determined by gel electrophoresis under denaturing conditions. Analysis of the trophoblastic layer primarily disclosed the presence of several additional sugar-binding proteins (lectins) in comparison to full-term placenta. The presence and developmental changes of such endogenous sugar receptors may lead to specific carbohydrate-protein interactions of physiological significance with similarly developmentally regulated carbohydrated portions of glyco-conjugates, already detected in human placenta by plant lectins.     

C32 human melanoma cell endogenous lectins: characterization and implication in vesicle-mediated melanin transfer to keratinocytes.

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.     

Localization of endogenous lectins in normal human breast, benign breast lesions and mammary carcinomas

Specific antisera against three mammalian beta-galactoside-specific lectins of apparent molecular weights 14.5 kDa, 18 kDa and 29 kDa have been used to localize these lectins in normal breast, and in benign and malignant mammary lesions. In normal breast tissue discrete localization of two lectins (Mrs 14.5 kDa and 18 kDa) was demonstrated in fibroblasts, smooth muscle cells, myoepithelial cells and capillary endothelium. Extracellular localization of one lectin (Mr 14.5 kDa) in collagen was apparent. The third lectin (Mr 29 kDa) labelled preferentially luminal cells and their secretory product. Two benign tumours (an analyzed fibroadenoma and a papilloma) revealed strong staining with two lectins (Mrs 18 kDa and 29 kDa). Of the 24 mammary carcinomas examined, the lectin (Mr 14.5 kDa) was expressed by only occasional tumour cells, the lectin (Mr 18 kDa) occurred in many tumour cells and the lectin (Mr 29 kDa) labelled tumour cells in nearly all cases. The expression of these beta-galactoside-specific endogenous lectins therefore appears to be regulated differently in normal breast compared with mammary tumours.     

Purification and characterization of beta-galactoside-binding proteins from Caenorhabditis elegans.

Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble beta-galactoside-binding lectins which had previously been only found in vertebrates.     

Evidence that Caenorhabditis elegans 32-kDa beta-galactoside-binding protein is homologous to vertebrate beta-galactoside-binding lectins. cDNA cloning and deduced amino acid sequence.

We have cloned a full-length cDNA for a beta-galactoside-binding protein with a relative molecular mass of 32 kDa (32-kDa GBP), recently purified from a nematode, Caenorhabditis elegans (Hirabayashi, J., Satoh, M., Ohyama, Y., and Kasai, K. (1992) J. Biochem. 111, 553-555). The clone contained a single open reading frame encoding 279 amino acids, including the initiator methionine. Significant sequence homology to metal-independent beta-galactoside-binding lectins (25-30% identities), which had previously been found only in vertebrates, was observed. Moreover, the nematode 32-kDa GBP proved to have a unique polypeptide architecture; that is, it is composed of two tandemly repeated homologous domains, each consisting of about 140 amino acids. The internal homology was about 32%. Thus, this protein is constructed with a duplicated fundamental unit which is similar to the subunit of vertebrate 14-kDa lectins. In spite of the extreme phylogenic distance between nematodes and vertebrates (divergence greater than 6 x 10(8) years ago), both of the two repeated domains of the nematode 32-kDa GBP retained most of the amino acid residues conserved in vertebrate lectins. This means that members of the metal-independent animal lectin family are distributed much more widely than had been believed: from nematodes to vertebrates. The implication is that proteins belonging to this family have fundamental roles which are not restricted to vertebrates but are common to almost all animals.     

Two novel lectins from Parkia biglandulosa and Parkia roxburghii: isolation, physicochemical characterization, mitogenicity and anti-proliferative activity

Two mannose/glucose specific seed lectins were isolated from Parkia biglandulosa and Parkia roxburghii and were characterized w.r.t various physicochemical properties. Unlike other Parkia lectins a comparison of native and subunit molecular mass showed that both Parkia lectins were heterotetramers. Parkia biglandulosa lectin was found to be T-cell mitogen as revealed by IL-2 bioassay. These lectins showed anti-proliferative effect on two murine macrophage cancer cell lines i.e. P 388DI (50%) and J774 (70%). In addition Parkia roxburghii also inhibited proliferation of HB98 (65.47%), a B-cell hybridoma cell line.     

Isolation and characterization of leukosialin, a major sialoglycoprotein on human leukocytes

A major sialoglycoprotein (previously called gp105) on the human erythroleukemic cell line K562 was purified, and specific antibodies were raised in a rabbit. A number of different hematopoietic cell lines belonging to erythroid, myeloid, T-lymphoid, and B-lymphoid cell lineages were found to possess glycoproteins that were immunoprecipitated by these antibodies. However, the apparent molecular weights differed between cell lines, ranging from 113,000 to 150,000. In almost all cases, the immune precipitated molecule corresponded to the major sialoglycoprotein of the respective cell. Pulse-chase experiments showed that all cells produced an early precursor form of the molecule of 54 kDa, which was susceptible to endo-beta-N-acetylglucosaminidase H to give an apoprotein of 52 kDa. Neuraminidase treatment of the mature forms resulted in a characteristic decrease of the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (apparent molecular weights from 150,000 to 183,000). Amino acid analysis of the glycoprotein isolated from HL-60 cells showed a high content of serine, threonine, and proline, and the carbohydrate composition was compatible with the presence of a large number (approximately 90) of O-linked carbohydrate chains. The name leukosialin is proposed for this sialoglycoprotein, which seems to be widely distributed, but differently glycosylated, on leukocytes with diverse functions. In the following paper (Carlsson, S.R., Sasaki, H., and Fukuda, M. (1986) J. Biol. Chem. 261, 12787-12795), we demonstrate that the structures of O-linked oligosaccharides vary significantly depending on the cells from which leukosialin was isolated.     

Oncogenes and expression of endogenous lectins and glycoconjugates

Summary— It is now well established that malignant transformation of eucaryotic cells is concomitant with typical alterations of glycosylation and the expression pattern of endogenous lectins. In parallel, oncogene transfection studies revealed a correlation between the expression of some of these genes, the transformed state and perhaps metastasis. These observations lead to the idea that oncogenes may control the expression of enzymes involved in the biosynthetic pathway of cell membrane glycoconjugates and the expression of endogenous lectins. Indeed, several contributions have shown that cells upon transfection with activated oncogenes of the ras family become invasive and/or metastatic and have their membrane glycoproteins modified. Information on the molecular mechanism of this postulated oncogene regulation is still lacking. Because of the diversity of the functions of oncogene-encoded proteins, further experiments dealing with other activated oncogenes may help in deciphering the regulation of expression of glycoconjugates and endogenous lectins together with their functions.     

Distribution and molecular evolution of rhamnose-binding lectins in Salmonidae: isolation and characterization of two lectins from white-spotted Charr (Salvelinus leucomaenis) eggs

L-Rhamnose-binding lectins were isolated from white-spotted charr (Salvelinus leucomaenis) eggs to understand the distribution and molecular evolution of the lectins in Salmonidae. Only two L-rhamnose-binding lectins, named WCL1 and WCL3, were isolated from white-spotted charr eggs, though three lectins, named STL1, STL2, and STL3, had been obtained from steelhead trout (Oncorhynchus mykiss) eggs. The cDNAs of WCL1 and WCL3 included 1,245 and 838 bp nucleotides with open reading frames of 933 and 651 nucleotides, respectively, and encoded for the complete amino acid sequences of mature proteins consisted of 288 (WCL1) and 195 (WCL3) residues, and signal sequences of 23 and 22 residues, respectively. WCLs were composed of three (for WCL1) or two (for WCL3) tandemly repeated homologous domains, which consisted of about 95 amino acid residues, and showed 91 and 93% sequence identities to STL1 and STL3, respectively. The mRNAs of WCL1 and WCL3 were detected exclusively in liver and ovary, respectively, however, neither a protein nor mRNA corresponding to STL2 could be identified in white-spotted charr. The phylogenetic tree of the sequences encoding carbohydrate recognition domains of 7 lectins from 4 species shows 5 functional clusters and their evolutional process. These results indicate that multiple L-rhamnose-binding isolectins have diverged by gene duplication and exon shuffling to play various biological roles in each species.     

Cloning, isolation and characterization of human tumor in situ monoclonal antibodies

A bacterially expressed human antibody (Ab) library (diversity approximately 10(5)) was generated from tumor-infiltrating B lymphocytes present in tissue isolated from a colon tumor. Immunoglobulin (IgG) heavy and light chain variable regions were amplified without isolating or enriching B cells, cloned into a phage-expression vector, and soluble antigen-binding fragment (Fabs) from >10(5) members of the library were screened rapidly by two distinct and complementary methodologies. In the first approach, soluble Fabs were screened by enzyme-linked immunosorbent assay (ELISA) on tumor cell monolayers. Alternatively, tumor cell surface antigens were selectively biotinylated with a plasma membrane-impermeable reagent, solubilized with non-ionic detergent, and were used to screen >10(5) members of the Ab library by capture lift. Reactive Fabs were partially characterized for tumor cell specificity and cross-reactivity, resulting in the identification of multiple Abs that bind cultured tumor cells but not normal human fibroblasts. The Fabs clustered into at least three distinct epitope specificity groups based on multiple criteria, including differential reactivity on two tumor cell lines and distinct antigen recognition patterns on western blot and immunoprecipitation. Moreover, DNA sequencing of the Ab variable regions demonstrated that the majority of the tumor-reactive Fabs were distinct and substantially different from the corresponding most homologous Ab germline gene. The relatively small size of the tumor-derived library allowed direct screening of soluble Fab of every member of the library, permitting the characterization of multiple human monoclonal antibodies (mAbs) that might not be discovered using alternative approaches, such as hybridoma technology or phage-display.     

Immunological cross-reactivity between beta-galactoside-binding lectins of vertebrates. Possible relationship of antigenicity to oligomeric structure

Structural relationships among five beta-galactoside-binding lectins isolated from human, mouse and chick were studied using immunochemical methods. The lectins examined were human placenta lectin with a 14-kDa subunit (human 14K lectin), two types of mouse lectin (mouse 15K and mouse 16K lectin), and two types of chick lectin (chick 14K and chick 16K lectin). Five polyclonal antibodies raised against these lectins were used. Antibody to human 14K lectin cross-reacted with mouse 15K and chick 14K lectins. Antibodies to both mouse 15K and chick 14K lectins cross-reacted with human 14K and chick 16K lectins. Antibody to chick 16K lectin cross-reacted with mouse 15K lectin. An immunological relationship was not found between human 14K and chick 16K lectins, or between mouse 15K and chick 14K lectins. Mouse 16K lectin did not show any immunological relationship with any of the other lectins. A monoclonal antibody raised against chick 14K lectin cross-reacted with chick 16K lectin. These results cannot be explained simply in terms of phylogenic distance but suggest that vertebrate beta-galactoside-binding lectins can be classified into two structural groups on the basis of their antigenicities. One group, which is characterized as a monomer type, includes human 14K and chick 14K lectins. The other group, which is characterized as a dimer type, includes mouse 15K and chick 16K lectins.     

Purification and characterization of beta-galactoside-binding lectin from chick embryonic skin

Lectin activity was found in tarsometatarsal skin of chick embryo. It was specific for beta-linked galactosyl residues and required a thiol-reducing agent for hemagglutination activity. The lectin was extracted from dermis and epidermis (skin) with lactose and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. Examination of their biochemical properties showed that although dermis and epidermis develop from different origins, they contain the same lectin. The apparent subunit Mr of lectin was 14000 and its isoelectric point was 7.0. Under non-dissociating conditions, the lectin exists mainly as a dimer. Radioimmunoassay showed that this skin-type lectin is present in many tissues including skin, muscle, bone, eye, heart, liver and brain at various developmental stages. A wide distribution and a marked change in its content during development strongly suggest that the lectin might have a fundamental role in cellular function, embryonic development and tissue differentiation.