Gerhard Uhlenbruck

Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. I. Histochemical distribution of lectin binding sites in normal alimentary tract as well as in benign and malignant gastric neoplasms

Labeled lectins with binding specificity to the hexose components of mucus glycoproteins (HPA, RCA I, PNA, Con A, WGA, and UEA I) were used to demonstrate structural differences in the glycoprotein composition of various cell types of the normal, benign and malignant gastrointestinal mucosa. While in the RCA I, UEA I, and WGA binding of normal mucus secreting cell types only quantitative differences were observed, the mucus in the surface epithelial cells of gastric mucosa and in the colonic goblet cells was characterized by the absence of PNA, Con A, and PNA, HPA binding sites, respectively. These lectins, however, showed a strong binding to the supranuclear, Golgi-region in the undifferentiated or activated forms of these cells. Even the staining intensity of the luminal membrane surfaces of the non mucinous parietal and chief cells was often stronger by PNA, HPA, and RCA I lectins than that of the mucus secretions in the highly differentiated mucus cells. These results indicate the existence of either heterogeneous glycoprotein components or mucus molecules with variations in the degree of glycosylation of their oligosaccharide chains in the different cells. The latter seems more likely since in benign and malignant alterations lectin binding sites appear in great density, which were found to be characteristic of the undifferentiated mucus cells or for the non mucinous cells of the normal gastric mucosa. Similarly in some gastric cancers which do not stain with the periodic acid-Schiff reaction at all, large amount of free or neuraminic acid substituted PNA binding sites can be detected.

Invertebrate Immunity: Another Viewpoint

All vertebrates and invertebrates manifest self/non‐self recognition. Any attempt to answer the question of adaptive significance of recognition must take into account the universality of receptor‐mediated responses. These may lake two forms: (1) rearranging, clonally distributed antigen‐specific receptors that distinguish in the broadest sense between self and non‐self, and non‐self A from non‐self B, latecomers on the evolutionary scene; (2) pattern recognition receptors, the earliest to evolve and still around, necessitating the requirement for induced second signals in T‐ and B‐cell activation. Either strategy need not force upon invertebrates the organization, structure and adaptive functions of vertebrate immune systems. Thus, we can freely delve into the unique aspects of the primitive immune mechanisms of invertebrates. In contrast, using the opposite strategy which is still problematic, i.e. linking invertebrate and vertebrate defence, seems to give us an approach to universality that might eventually reveal homologous kinship.

Inhibition of liver metastasis in mice by blocking hepatocyte lectins with arabinogalactan infusions and d-galactose

SummaryAccording to our hypothesis, organ-specific lectins (e.g., the d-galactose-specific hepatic binding protein) play an important role in the organ location of metastatic malignant cells. The rapid clearance and uptake by the liver of tritiated α-acid-(asialo)glycoprotein from the circulation of Balb/c mice was markedly delayed after preinjection of d-galactose or arabinogalactan. The preinjection (1h) and regular application (for 3 days after tumor cell inoculation in Balb/c mice) of the receptor blocking agents d-galactose and arabinogalactan prevented the settling of sarcoma L-1 tumor in the liver completely, but did not influence the settling in the lung. Other galactans, dextrans, and phosphate-buffered saline showed no effect. Therefore, when lectins were blocked with competitive-specific glycoconjugates, colonization was prevented.

Characterization of the oligosaccharide side chain of apolipoprotein C-III from human plasma very low density lipoproteins

Apolipoprotein C-III1 and apolipoprotein C-III2 each contain one oligosaccharide side chain, bound O-glycosidically to threonine in position 74 of the amino acid sequence. The studies reported in this paper characterize these alkali labile oligosaccharides, thereby demonstrating the complete structure of apolipoprotein C-III. Monosaccharide analysis revealed the following sugar composition: D-galactose/N-acetyl-D-galactosamine/sialic acid 1 : 1 : 1 and 1 : 1 : 2 for apolipoprotein C-III1 and apolipoprotein C-III2, respectively. Treatment of desialylated apolipoproteins with alkaline borohydride released the reduced disaccharide beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol, which was detected by gas-liquid chromatography. Further studies employing periodate oxidation and Smith degradation indicated that the structure of the trisaccharide from apolipoprotein C-III1 was alpha-N-acetylneuraminyl-(2 leads to 3)-beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosaminitol. The tetrasaccharide structure from apolipoprotein C-III2 is made up of this trisaccharide plus one sialic acid residue linked to C6 of N-acetyl-D-galactosaminitol, as was shown by the assessment of chromogens formed upon alkaline degradation.

Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins.

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins:1)the size of the alditols varies from tri- to decasaccharides2)the core structure is of the ubiquitous type 23)the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAcβ(1–3)Gal (major) or GlcNAcβ(1–6)Gal units (minor).N-Acetylneuraminic acid on monosialylated (mucin- or glycopeptide-derived) and disialylated glycans (glycopeptide derived) is linked predominantly to position C-3 of galactose.

Alkali-labile oligosaccharides from glycoproteins of different erythrocyte and milk fat globule membranes

Phenol extraction of horse, sheep, cow, pig and human erythrocyte membranes and human milk fat globule membranes gave glycoprotein fractions, all of which were shown by gas chromatography to contain the reduced disaccharide beta-D-galactosyl (1-3)-N-acetyl-D-galactosaminital after treatment with alkaline borohydride. Cow and pig erythrocyte membrane glycoproteins were found however to contain much lower amounts than the erythrocyte membrane glycoproteins of the other species tested. After gel filtration, a tetrasaccharide was isolated from horse and sheep glycoproteins containing the disaccharide plus two molecules of sialic acid. Periodate oxidation together with paper chromatography of alkaline degraded fragments showed these two molecules of sialic acid to be linked to positions C3 and C6 of the galactosyl and N-acetylgalactosamine residues respectively. Evidence was obtained for a similar structure from pig and cow erythrocyte glycoproteins and human milk fat globule membrane glycoproteins although the complete structure was not elucidated. In all native glycoprotein fractions, the unsubstituted disaccharide beta-D-galactosyl (1-3)-N-acetyl-D-galactosamine was found to be present to different extents. Haemagglutination inhibition tests against human anti-T serum, Arachis hypogoea and Vicia graminea by desialylated glycoproteins showed the presence of the T-antigen, confirming the chemical findings. Inhibition was found to be proportional to the chemically detected amounts of disaccharide in each fraction. Evidence for a second carbohydrate chain in horse, sheep and human erythrocyte glycoproteins with a sialic acid substituted N-acetylgalactosamine residue as the terminal sequence was obtained using the agglutinin from Helix pomatia.

Importance of lectins for the prevention of bacterial infections and cancer metastases.

Adhesion of bacteria and of metastasizing tumour cells have much in common, especially the participation of lectins in this process. In the future it might be possible to inhibit the metastatic process and bacterial adhesion by blocking with lectins specific for appropriate (oligo) saccharides or glycoconjugates. Initial clinical trials are very promising.

Structural studies on oncofetal carbohydrate antigens (Ca 19-9, Ca 50, and Ca 125) carried by O-linked sialyl-oligosaccharides on human amniotic mucins☆

Mucins were extracted from human amniotic fluid in the presence of 45% vol. phenol and separated from the bulk of smaller-sized glycoproteins by exclusion on Sephacryl S400. The mucin-fraction FW, which still contained a minute proportion of mannose, strongly expressed oncofetal antigens recognized by monoclonal antibodies C 50, NS 19-9, OC 125, Leu M1, 49 H 8, and 115 C 2. The structures of the respective mucin-linked saccharides responsible for Ca 50-, Ca 19-9-, and Lea-related antigenic activities were analyzed before or after reductive beta-elimination from sialylglycoproteins, and purification of the derived alditols by gel-permeation chromatography on Bio-Gel P-4 or high performance liquid chromatography. Two ubiquitous (FW2, FW3) and three novel oligosaccharide alditols (FW5) were characterized by f.a.b.- and e.i.-m.s., combined with methylation analysis and chromium trioxide oxidation. The OC 125 epitope on mucin-carried O-glycans was destroyed during reductive cleavage of the saccharides, indicating a conformational involvement of the reducing terminal residue and its mode of conjugation to the protein. Exoglycosidase treatment of the mucin-bound antigen revealed that the epitope structure of OC 125 includes terminal beta-D-galactosyl groups, and terminal sialyl groups that are almost inaccessible to Vibrio cholerae sialidase digestion.

Investigation on extracellular slime substance produced by Staphylococcus epidermidis

The extracellular slime substance produced by Staphylococcus epidermidis was investigated. Slime production was assessed by bacterial agglutination in the presence of concanavalin A (Con A) or poly-L-lysine and by bacterial adherence to polyethylene. Media for slime production was optimized using these criteria. A phenol-saline extract of crude slime was separated into four fractions on a DEAE-sepharose column. Total protein and sugar content and the monosaccharide constituents were determined. Crude slime and the phenol-saline extract showed a strong precipitation reaction with Con A and poly-L-lysine (double diffusion). Fractions I and II containing mannose as the most abundant sugar reacted with Con A and two other mannose-specific lectins (Lens culinaris, Pisum sativum). This reaction could be inhibited by mannose. Fractions III and IV were precipitated by poly-L-lysine, probably due to a reaction with glucuronic acid which was only present in these fractions. Precoating of polyethylene with crude slime, phenol-saline extract and fractions III and IV resulted in a marked inhibition of attachment of staphylococcal cells. Production of the extracellular slime substance was completely inhibited by subinhibitory concentrations of the glycosylation inhibitor tunicamycin, whereas penicillin had no influence. Extracellular slime substance produced by S. epidermidis seems to be a complex of glycoconjugate character and plays an important role in the attachment to synthetic polymers. The production of slime by staphylococci can be easily determined using mannose specific lectins and poly-L-lysine.

Heterogeneity of human red cell membrane sialoglycoproteins

SummaryDiscontinuous sodium dodecylsulfate polyacrylamide gel electrophoresis (disc SDS-PAGE) followed by periodic acid/Schiff staining reveals the presence of six sialoglycoprotein bands in human red cell membranes or glycoprotein preparations therefrom. In agreement with previous investigations it is shown that PAS-1 and PAS-2 (mol. weight 37 000) are different forms of the same molecule (MN glycoprotein). Using separation of glycoproteins by the system ofWeber andOsborn and reelectrophoresis of gel slices by disc SDS-PAGE it is demonstrated that the minor component C (mol. weight 41 000) represents the dimeric form of PAS-3 (Ss glycoprotein). Band B corresponds to an aggregate of PAS-3 and PAS-2 and/or the trimer of PAS-3 with possible differences between extracted glycoproteins and those present in the membrane. The minor component D (mol. weight 35 000) is, as far as could be elucidated, not involved in aggregation phenomena. Some technical problems of glycoprotein fractionation by SDS-PAGE and the remarkable effect of phosphate buffers on the glycoprotein pattern are discussed.ZusammenfassungDurch diskontinuierliche Natrium-Dodecylsulfat Polyacrylamid Gel Elektrophorese (disc SDS-PAGE) und anschlie\ende PerjodsÄure/Schiff-FÄrbung lassen sich sechs Sialoglykoproteinbanden in den aus menschlichen Erythrozytenmembranen extrahierten Glykoproteinpreparationen feststellen. In übereinstimmung mit früheren Untersuchungen wird gezeigt, da\ die Banden PAS-1 und PAS-2 (Mol.-Gewicht 37 000) verschiedene Formen desselben Moleküls (MN Glykoprotein) darstellen. Durch Auftrennung von Glykoproteinen mit dem System vonWeber undOsborn und anschlie\ende Reelektrophorese von Gelstücken mit disc SDS-PAGE wird nachgewiesen, da\ die Komponente C (Mol.-Gewicht 41 000) das dimere PAS-3 (Ss Glykoprotein) darstellt. Die Bande B entspricht einem Aggregat aus PAS-2 und PAS-3 und/oder dem Trimer von PAS-3. Möglicherweise bestehen Differenzen zwischen extrahierten Glykoproteinen und solchen in der Membran. Die Komponente D (Mol.-Gewicht 35 000) ist, soweit sich klÄren lie\, nicht an AggregationsphÄnomenen beteiligt. Einige technische Probleme der Glykoproteinauftrennung mit SDS-PAGE und der bemerkenswerte Effekt von Phosphatpuffern auf das Glykoproteinmuster werden diskutiert.