Lea Activity Research Articles

Changes of Serological Activity by α-L-Fucosidase Isolated from Streptococcus sanguis ATCC 10557

alpha-L-Fucosidase isolated from the growth culture of Streptococcus sanguis ATCC 10557 acted on H- and Leb-blood group substances in porcine gastric lining, human gastric lining, human ovarian cyst fluid and human whole saliva, with consequent loss of H- and Leb -activities and a concomitant increase of Lea activity.

Characterization of a human intestinal fucolipid with blood group Lea activity.

A fucolipid that carried human blood group Lea activity was isolated from human small intestine. It contianed fucose, galactose, N-acetyl glucosamine, glucose, and ceramide in a molar ratio of 1:2:1:1:1. After periodate oxidation only 1 molecule of galactose and the N-acetylglucosamine remained. Permethylation of the lipid gave derivatives of a terminal fucose and galactose residue together with 2,4,6-tri-O-methylgalactose and 2,3,6-tri-O-methylglucose. After removal of fucose the lipid could be converted to a ceramide trihexoside with beta-galactosidase, and this, in turn, to ceramide lactoside by the action of beta-N-acetylhexosaminidase. Both enzymes converted the defucosylated derivative to a ceramide monohexoside. The methylated and the methylated and reduced derivatives of the intact lipid gave ions in mass spectrometry for a terminal hexose and deoxyhexose, a terminal trisaccharide of hexose, deoxyhexose and N-acetylhexosamine, and terminal tetra-and pentasaccharides. Ceramide fragments characteristic of hydroxy fatty acids with 16, 22, 23, and 24 carbons were found together with those of phytospingosine as the major long chain base. On the basis of these results and the immunologic activity of the fucolipid, the following structure is proposed: betaGal (1 leads to 3)betaGlcNAc (1 leads to 3)betaGal (1 leads to 4)Glc-ceramide alphaFuc (1 leads to 4).

The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by density-gradient ultracentrifugation.

1. Equilibrium density-gradient ultracentrifugation in caesium salts was used in two stages in the isolation and subfractionation of the glycoprotein component from a human ovarian-cyst fluid. The eight main subfractions thus obtained were the subject of detailed physicochemical characterization. 2. The fractions were unimodal in buoyant-density distribution, but had discrete rho(0) values ranging from 1.31 to 1.35. 3. Weight-average molecular weights and sedimentation coefficients decreased regularly with decreasing density of the fraction, whereas the partial specific volumes and selective solvation parameters increased. The latter behaviour correlates well with the increasing peptide content of the lighter fractions. 4. The fractions exhibited a range of analytical composition, although all were within the limits previously observed for blood-group substances of Le(a) specificity. All fractions had approximately equal Le(a) activity. The peptide content varied systematically from 7% for the densest fraction to 15% for the lightest, but the relative distributions of the amino acids remained essentially constant throughout the series. In particular, serine plus threonine plus proline made up about 50% of the peptide content of all the fractions. Fucose, galactose and N-acetylglucosamine contents decreased with increasing peptide content of the fractions, but N-acetylgalactosamine and sialic acid exhibited the opposite trend. Molar ratios of N-acetylgalactosamine to the sum of serine and threonine remained essentially constant at 0.8-0.9, implying a high degree of glycosylation of all the molecules, but the ratio of N-acetylglucosamine to N-acetylgalactosamine decreased steadily with increasing peptide content, suggesting the presence of oligosaccharide side chains of various lengths. The results are discussed in terms of the accepted structure of glycoprotein molecules. 5. Experiments on the glycoproteins extracted with phenol from the same cyst fluid have confirmed that equilibrium centrifugation in caesium salts does not remove any non-covalently bound protein nor cause any changes in the tertiary structures of these glycoprotein molecules.

The radioimmunoassay of serum and salivary blood group A and Lea glycoproteins.

Summary. A radioimmunoassay was developed for the detection of A and Lea blood group activities in glycoprotein fractions extracted with perchloric acid (PCA) from normal serum. The technique was also applied to studies of salivary blood group glycoproteins.Blood group A activity was detected in all group A sera tested. Higher levels of A activity were found in sera from group A1 than from A2 donors, and sera from both group A1 and A2 secretor donors gave higher levels of A activity than sera from non‐secretor donors. No difference was found in the levels of A activity in group A1 and A2 secretor salivas but only small numbers were assayed. Parallel radioinimunoassay inhibition lines, indicating immunological identity, were obtained with PCA‐extracts of A1 and A2 secretor plasma and with extracts of A1 and A2 secretor salivas.Lea activity was detected in all sera tested. The levels of Lea activity were higher in sera from Le(a + b ‐) donors than from h(a ‐ b +) or Le(a ‐ b ‐) donors and were unaffected by ABO group. Radioimmunoassay inhibition lines indicated qualitative differences in the Lea antigen in salivas from secretors and non‐secretors, consistent with a lower density of Lea antigen on salivary glycoprotein from secretor donors.

Quantitative studies on salivary blood group substances. 3. In identical twins.

Abstract With the use of a quantitative automated method, the concentrations of blood group substances A, B, H and Lea have been measured in salivas from 100 pairs of identical twins. Titers of Leb substance were determined manually. When the sample is grouped according to types, highly significant independent interactions of ABO blood types on the concentration of H and Leb activity and of secretor type on Lea activity are shown. It is also brought out in discussion that concentrations of salivary A and B substances are significantly higher in Lewis‐negative individuals. However, no influence of ABO type is shown on Lea activity in cell types Le(a+b‐) or Le(a‐b+). Highly significant allelic interactions are shown to influence the concentrations of A and B substances in A and B types and A subtypes. Thus, a major contribution to good intra‐pair correlations in the concentration of blood group activity in related individuals, particularly in identical twins, will be made by their similarity in ABO, secretor and Lewis types. However, when analyses of the twin data are restricted to pairs of the same ABO, secretor and Lewis types, intra‐pair correlations for A, B, H, Lea and Leb substance are highly significant in the majority of groupings except in the Lewis‐positive nonsecretors. This concordance is shown to be independent of sex, age or environment. It is concluded, therefore, that the concentration of salivary blood group substances A, B, H, Lea and Leb in secretors is under close genetic quantitative regulation. The suggestion is made that a series of quantitative Se alleles could be the source of such control.

Studies on the secretion of blood group substances. II. Observations on the red cell phenotype Le(a-b-x-).

Abstract Salivas from 35 type Le(a‐b‐x‐) subjects were tested for Lea,Leb, Lex, A, B and H substances by both manual and automated quantitative hemagglutination inhibition methods. For comparison, similar data were gathered from 226 Lewis positive adults. Among the 13 Ma‐bx‐) nonsccretors, 7 had small quantities of salivary Lea activity and 11 had low concentrations of salivary Lex by manual methods. None showed any salivary Lebactivity. By the automated method, low concentrations of Leaactivity were demonstrable in 12. Among the 22 Ma‐b‐x‐) ‐tors, 6 had minute quantities of salivary Lewis activity, mainly Leb. The Leb activity could not represent cross reactivity with H substance because most of those subjects with the highest H activities had no Leb activity.Our quantitative studies show, when salivary Lewis activity is present in individuals whose red cells era type Ma‐bx‐), that the activity is much lower in comparison with individuals whose red cells are Lewis positive. Thus, based on quantitative salivary studies, it is possible to distinguish between Lewis positive (Len and Lewis negative (le/le) individuals.These observations suggest that the le gene is not a completely inert allele but that it governs the biosynthesis of serologically active substances. The Ze gene can be regarded as an inefficient Le which produces exactly the same products in a qualitative way as the Le gene but quantitatively far less. Another possibility is, as recently suggested, that the le gene actually governs the biosynthesis of distinct products Lec and Led and that salivary Lea and Leb activities among Lewis negative subjects represent cross reactivity with these products.

Intestinal Sphingoglycolipids with A and Lea Activity from Humans and A, H-Like and Leb-Like Activity from Dogs

Abstract Glycolipids containing fucose (fuco-lipids) have been shown to constitute an individual specific class of compounds both in humans and dogs. Since only trace amounts of some fuco-lipids can be isolated, immunologic techniques are invaluable in identifying these compounds and giving some insight into their structure. By using human and rabbit antisera and ulex lectin with precipitation and agglutination techniques, fuco-lipids isolated from human small intestine were shown to have human A and Lea blood group activity. Fuco-lipids isolated from dog small intestine had human A, H-like and Leb-like blood group activity. With fluorescein-labeled rabbit antisera it was demonstrated that Forssman activity was associated with the lamina propria whereas Leb-like activity was in the glandular epithelium of the small intestine.

COMPARISON OF HUMAN GASTRIC CANCER AND GASTRIC LINING MUCOPOLYSACCHARIDES

The specimens removed surgically for gastric cancer were divided into tumor tissues and tissues considered to be normal, and the normal tissues were further divided into the mucosal linings and underlying tissues (muscularis mucosae). Mucopolysaccharides of these three portions of each specimen were extracted with 90% phenol after peptec hydrolysis of the tissues. Mucopolysaccharides were found as a phenol-insoluble residue and phenol soluble fraction precipitated with alcohol at a final concentration of 10%.There were certain differences between phenol-insoluble fraction of gastric cancer and normal gastric lining in blood group activities. Some of cancer mucopolysaccharides from secretor patients had weaker Leb activity than Lea activity than that of mornal gastric lining of same patient. It suggests that in the gastric cancer. tissues the blood group substances remain underdeveloped stage of their biosynthesis. Quantitative analytical results showed that some of phenol-insoluble fractions from gastric cancer had higher contents of total sugar and hexosamine than do same fraction of normal gastric linings. Some of phenol-insoluble fraction from gastric cancer contained glucose as a sugar components in addition to galactose, fucose and two amino sugars, glucosamine and galactosamine which were components of blood group substances.

Immunologic Relationship between Blood Group Substances and a Fucose-Containing Glycolipid of Human Adenocarcinoma

Summary A fucose-containing glycolipid isolated from human adenocarcinoma tissue was found to lack blood group A or B activity regardless of the blood type of the tissue donor. The tumor glycolipid had weak H and moderate Lea activity. Tumor glycolipid was precipitated by type XIV pneumococcal polysaccharide antiserum only after mild acid hydrolysis. Antisera to tumor glycolipid agglutinated erythrocytes of type A more strongly than erythrocyte of type B, AB, or O. Furthermore, tumor glycolipid and type A glycolipid inhibited the agglutination of tumor cells by a certain phytoagglutinin more strongly than other glycolipids tested. Thus blood group A substance or erythrocytes appear to contain increased amounts of a hapten structure similar to or identical with the carbohydrate moiety of tumor glycolipid. It is suggested that tumor glycolipid results from an aberration in synthesis of glycolipids possessing blood group activity.

STUDIES ON Lea-DECOMPOSING ENZYME

The chemical action of purified Lea decomposing enzyme preparation from Bac. cereus on the water-soluble Lea and O(H) substances has been examined. Besides β-galactosidase, this enzyme preparation contains α-fucosidase which is different from that in O(H) and Leb enzyme preparation from Bac. fulminans or Bac. cereus O(H).1) Lea enzyme preparation acts on Lea substance to leberate about 34% of the original fucose and 32% of the original galactose as simple sugars.2) Lea enzyme preparation acts on O(H) substance to liberate fucose and galactose as simple sugars amounting to 3% and 19% of the original quantities respectively. Subseqent treatment with O(H) and Leb enzyme preparation from Bac. fulminans appears Lea activity and liberates from the Lea enzyme treated substance 32% of the original fucose and 2% of the original galactose as simple sugars. The third treatment with Lea enzyme preparation destroys newly developed Lea activity and liberates fucosc and galactose as simple sugars amounting to 28% and 8% of the original quantities respectively.

CHANGES OF LEWIS BLOOD GROUP SUBSTANCE INDUCED BY ENZYMES

1. A crude enzyme preparation from Bac. fulminans or from Bac. cereus O(H) contained O(H) -and Leb-decomposing enzymes, and when applied on an O(H), Le (a±b+) substsnce, destroyed O(H) and Leb activity together with enhancing Lea activity. But when applied on an O(H), Le (a±b-) substance, it only destroyed O(H) activity without enhancing Lea activity, However, when the O(H), Le (a±b+) substance was incubated with purified preparation from Bac. fulminans, which contained only the O(H) -decomposing enzyme, abolishment of O(H) activity alone was observed, but not enhancement of Lea activity. These results seem to indicate that from a precursor substance are individually developed O(H) and Lea substance, and on the basis of Lea subsfance is formed Leb substance.However, when the crude enzyme preparation from Bac. fulminans or from Bac. cereus O(H) was applied on OLe (a-b+) red cells, it was difficult to recognize appearance of Lea activity, though O(H) and Leb activity were abolished.2. When the A-decomposing enzyme from Cl. tertium and the B decomposing enzyme from Cl. maebashi were applied on A and B substance or red cells with Lebactivity respectively, slight increase in Leb activity was observed together with enhancement of O(H) activity, When AB substance or AB red cells were incubated with both the A- and B-decomposing enzymes, these were transformed into a substance or red cells having O(H) activity nearly to the same degree as that of O(H) substance or O red cells.

STUDIES ON Lea-DECOMPOSING ENZYME

1. A strain of Bacillus cereus was isolated from the soil which produced an enzyme acting on water-soluble Lea substance and group OLe (a+b-) red cells. The enzyme preparation also contains A-decomposing enzyme, but not O(H) -, Leb-and B-decomposing enzymes. Decomposition of Lea activity of Lea substance and Le (a + b-) red cells with this enzyme resulted in the enhancement of the cross-reactivity of Lea substance and Le (a+b-) red cells with anti-pneumococci Type XIV chicken serum.2. These enzymes were found in a fraction which precipitated at 30-60% ammonium sulfate saturation of bacterial autolysate. By means of DEAE cellulose column chromatography, the Lea-decomposing enzyme could be separated from the A-decomposing enzyme. The optimal pH of these enzyme action ranged 6.8-7.2, the optimal temperature 30-37°C and the enzymes were inactivated by heating at 70°C for 5 minutes.3. The action of these enzymes were inhibited by such metal salts as copper sulfate, mercuric chloride and strontium nitrate. The enzyme action to decompose Lea substance was inhibited strongly by L-fucose, D-galactose and lactose, and slightly by β-ethyl-N-acetyl-D-glucosaminide and D-glucosamine. The results of the inhibition of enzyme action by sugars indicate that the structure of L-fucose and β-linked D-galactose combined with β-D-N-acetylglucosamine is important as antigenic determinant of Lea specificity. The action of A-decomposing enzyme was inhibited strongly by N-acetyl-D-galactosamine and slightly by D-galactosamine.

On a Lea Substance-Decomposing Enzyme. I

1. A strain of Bacillus cereus, which produced Lea-decomposing enzyme, was isolated from the soil. This enzyme, when applied to Le (a+b-) red cells or water-soluble Lea substance, decomposed it to abolish Lea activity, and enhanced its cross-reactivity with anti-pneumococci Type XIV chicken serum. In addition to this enzyme, the strain produced A-decomposing enzyme. By means of DEAE cellulose column chromatography, Lea-decomposing enzyme could be separated from A-decomposing enzyme. These enzymatic activities were found in a fraction that precipitated at 30-60% ammonium sulphate saturation of autolysate of the strain. Their optimal pH ranged from 6.8 to 7.2, and their optimal temperature ranged from 30° to 37°C. Treatment at 70°C for 5 minutes inactivated them.2. The actions of these enzyme preparations were slightly inhibited by copper sulphate, mercuric chloride and strontium nitrate. The action of the Lea-decomposing enzyme was also inhibited strongly by such sugars as L-fucose, D-galactose and lactose, and weakly by β-ethyl-N-acetyl-D-glucosamine and D-glucosamine, and action of the A-decomposing enzyme was inhibited strongly by N-acetyl-D-galactosamine, and weakly by D-galactosamine.

Failure to elicit a Delayed Hypersensitivity Reaction against Blood Group Substance from Erythrocytes

IN some recent work1, we have explored the antigenicity in guinea pigs of the secreted human blood group substances A (ref. 2) and Lea (ref. 3). There was a delayed hypersensitivity response to A and Lea, and to other human blood group substances, showing a very marked degree of cross-reactivity, irrespective of the immunizing antigen. This cross-reaction and other evidence were taken to indicate that the polypeptide part of blood group mucopolysaccharides was the entity chiefly concerned in producing and eliciting delayed hypersensitivity to these substances. The reaction of A and Lea substance with circulating antibody was much more specific than was the delayed hypersensitivity reaction. The small degree of cross-reactivity of circulating antibody could be attributed to a small amount of Lea activity in A substance. The more precise nature of the circulating antibody was explained in terms of a response to the specific polysaecharide entity of blood group substances. Thus, it was postulated that the secreted blood group substance macromolecule was capable of evoking separate immunological responses to its two main chemical components, the polypeptide and the polysaecharide.

The Production of Precipitating Antibody in Chickens to a Substance Present in the Fluids of Nonsecretors of Blood Groups A, B and O

Summary Substances have been isolated from the ovarian cyst of a woman of blood group A who is a nonsecretor of A substance, and from the saliva of a man of group B who is a nonsecretor of B substance. Injection of these materials into chickens resulted in the production of precipitating antibody. The antisera exhibited a specificity for the secretions and derivative substances of A, B and O nonsecretors; there was good correlation between Lea activity of the sample and its ability to precipitate the chicken antisera. It has not, however, proven possible to demonstrate experimentally prepared specific agglutinins for Le(a+) red cells nor can such cells absorb the precipitins from the chicken sera.