Localization of endogenous beta-galactoside-binding lectin in human cells and tissues.

A rabbit antiserum raised against the 14.5-kilodalton (kDa) subunit of human splenic galaptin was used to probe protein blots of several tissue extracts. For all tissues examined, the only immunoreactive species detected was a 14.5-kDa polypeptide. This antiserum and a rabbit antiserum raised against native lung galaptin were used in immunohistochemical assays to determine the localization of galaptin in selected tissues and cells. In normal colon, galaptin was found prominently in the basement membrane and in the stroma. The cytoplasm of epithelial cells stained lightly for galaptin whereas mucous granules and secreted mucin were uniformly negative for galaptin. Hemagglutination inhibition assays also failed to demonstrate an interaction between galaptin and mucin. Macrophages stained conspicuously for galaptin in colonic and cutaneous tissue as did some capillary walls. In cutaneous tissue, the extracellular matrix and hair follicle cells contained abundant galaptin. Galaptin was absent in basal cell carcinoma and associated stroma. Galaptin was found throughout the cytoplasm of carcinoma cells of gynecologic origin present in effusions. Protein blot analysis of extracts of extracellular matrix synthesized in vitro by endothelial cells confirmed the presence of galaptin in matrix. The results show that: (1) galaptin is variably expressed by different cells and tissues; (2) its cellular location is not restricted to the cell surface; (3) galaptin is not associated with normal mucin; (4) the extracellular matrix is a major site of galaptin deposition, and (5) some malignant tissue may be characterized by a deficiency of galaptin.

Galactoside-binding lectin in human tissues.

Lactose-inhibitable lectin activity has been analyzed by hemagglutination assay in a variety of human tissues and cells obtained at surgery and autopsy. The lectin activity was detected in surgically removed melanoma, sarcoma, colon carcinoma, breast carcinoma, adjacent non-malignant tissues, non-malignant tissues obtained at autopsy and in cells isolated from malignant effusions. Although, on average, malignant tissue had a higher hemagglutinating titer than non-malignant tissue, similar tissues from different individuals varied widely in their apparent lectin content. The lectin was isolated from lung by affinity chromatography and was found to have a native molecular mass of 31,000 daltons and a subunit molecular mass of 14,000 daltons. Utilizing rabbit anti-lung lectin serum in an immunohistochemical assay, the lectin was found to be distributed throughout the cytoplasm of lung epithelial cells. Ouchterlony immunodiffusion analysis confirmed the presence of this lectin in a variety of tissues and in some body fluids. In vitro metabolic radiolabelling experiments showed that the presence of lectin in tissues was most likely due to endogenous synthesis rather than absorption from body fluids. Lectin isolated from several tissues was found to bind to human buffy coat cell receptors.

In vitro synthesis of carbohydrate-binding proteins by human leucocytes.

A carbohydrate-binding protein (CBP) synthesized in vitro by normal human peripheral leucocytes was isolated by affinity chromatography on asialofetuin-Sepharose. The CBP was eluted with lactose and it had a native molecular mass of 15,500 daltons. Analysis by SDS-PAGE revealed a single polypeptide of 18,000 daltons. CBP synthesis was time dependent and cell concentration dependent. The CBP appeared to be both cell bound and secreted with the apparent amount secreted inversely proportional to cell concentration. CBP did not appear to be synthesized by T and B leukemic cell lines examined. Promyelocytic HL-60 cells, however, synthesized at least two lactose-eluted CBP's corresponding to native molecular masses of 28,000 and 19,500 daltons. SDS-PAGE analysis of radiolabelled HL-60 CBP's showed the presence of two polypeptides of MM 17,700 and 16,000 daltons suggesting that one of the CBP's was a dimer.

A study of the specificity of Bandeiraea simplicifolia lectin I by competitive-binding assay with blood-group substances and with blood-group A and B active and other oligosaccharides.

The specificity of Bandeiraea simplicifolia lectin I (BS I) has been studied by competitive-binding assays (CBA) using tritium-labeled human B and hog A substances. Blood-group B substances isolated from horse gastric mucosae and from human ovarian-cyst fluids were much better inhibitors of binding of tritiated blood-group B substance to insoluble BS I-Sepharose 2B than were human blood-group A substances from saliva and ovarian-cyst fluid. A and B active blood-group substances showed the same range of potency in inhibiting binding of tritium-labeled hog A substance to BS I-Sepharose 2B. CBA with BS I-Sepharose 2B, labeled human blood-group B substance, and human blood-group A and B active aligosaccharides separated the haptens into two groups differing in slope. Group 1, containing methyl alpha-D-GalNAcp, D-GalNAcp, and an A active pentasaccharide ARL 0.52, with 3, 19, and 25 nmol respectively needed for 50% inhibition of binding, has a lower slope than group 2, which contains alpha-D-GalNAcp-(1 leads to 3)-2-acetamido-2-deoxy-D-galactitol and p-nitrophenyl alpha-D-GalNAcp, with 3 nmol of each required for 50% inhibition of binding, as well as ten glycosides with terminal, nonreducing, alpha-linked D-Galp. The most potent inhibitors of this group were p-nitrophenyl alpha-D-Galp, alpha-D-Galp-(1 leads to 3)-D-Galp, alpha-D-Galp-(1 leads to 6)-D-Glcp, and methyl alpha-D-Galp, with 5, 7.4, 9.6, and 11 nmol respectively needed to inhibit binding by 50%. The difference in slopes was explainable in terms of a recent finding that BS I exists as a mixture of five isolectins composed of two subunits having different specificities; subunit A is most specific for alpha-linked, terminal, nonreducing D-GalNAcp, but it also reacts with alpha-linked, terminal, nonreducing D-Galp, whereas subunit B tends to be more specific for terminal, nonreducing, alpha-linked D-Galp.

Immunochemical studies on blood groups LXVI. Competitive binding assays of A1 and A2 blood group substances with insolubilized anti-A serum and insolubilized A agglutinin from Dolichos biflorus.

Competitive binding assays using 3H-labeled blood group A substance and insolubilized Dolichos biflorus lectin or human anti-A were carried out, measuring competition by blood group A1 and A2 glycoproteins, and by unabsorbed anti-A sera, and with these sera absorbed with the A1 and A2 glycoproteins. With Dolichos lectin specific for (formula: see text) A1 substances had about 11 times as many determinants as did A2 substances, but the slopes of the lines in the competitive binding assays were the same. With insolubilized anti-A, A2 substances gave lines of lower slopes. Although individual A1 populations varied in the amounts giving 50% inhibition in the assays, as did A2 substances, the slopes of the lines for the A1 substances were the same and always higher than the slopes of the lines for the A2 substances. Competitive binding assays with unabsorbed anti-A sera and with these sera absorbed with insoluble polyleucyl A1 and A2 substances showed that partial absorption of polyleucyl A1 substances left antibodies of lower slope in the supernate, whereas absorption with polyleucyl A2 substance left antibodies (anti-A1) having the same or an even higher slope than the unabsorbed sera. The findings indicate that human A1 and A2 glycoproteins differ in their determinants, and that A2 specificity is determined by the type 2 chain in which the A trisaccharide (formula: see text) is linked beta 1 leads to 4 to DGlcNAc, whereas the A1 specificity is determined by the type 1 chain in which this trisaccharide is linked beta 1 leads to 3 to DGlcNAc; most of the determinants in the glycoproteins have a second LFuc linked alpha 1 leads to 3 and alpha 1 leads to 4 to the DGlcNAc of the type 2 and type 1 chains, respectively.