Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane.

Porcine pancreatic α-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680-4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na(+)/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycan-specific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic α-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic α-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other.

Regulatory properties of vitronectin and its glycosylation in collagen fibril formation and collagen-degrading enzyme cathepsin K activity.

Vitronectin (VN) is a glycoprotein found in extracellular matrix and blood. Collagen, a major extracellular matrix component in mammals, is degraded by cathepsin K (CatK), which is essential for bone resorption under acidic conditions. The relationship between VN and cathepsins has been unclear. We discovered that VN promoted collagen fibril formation and inhibited CatK activity, and observed its activation in vitro. VN accelerated collagen fibril formation at neutral pH. Collagen fibers formed with VN were in close contact with each other and appeared as scattered flat masses in scanning electron microscopy images. VN formed collagen fibers with high acid solubility and significantly inhibited CatK; the IC50 was 8.1-16.6 nM and competitive, almost the same as those of human and porcine VNs. VN inhibited the autoprocessing of inactive pro-CatK from active CatK. DeN-glycosylation of VN attenuated the inhibitory effects of CatK and its autoprocessing by VN, but had little effect on acid solubilization of collagen and VN degradation via CatK. CatK inhibition is an attractive treatment approach for osteoporosis and osteoarthritis. These findings suggest that glycosylated VN is a potential biological candidate for CatK inhibition and may help to understand the molecular mechanisms of tissue re-modeling.

Pseudoproteoglycan (pseudoPG) probes that simulate PG macromolecular structure for screening and isolation of PG-binding proteins.

A proteoglycan (PG) monomer is a macromolecule consisting of one or more glycosaminoglycan (GAG) chains attached to a core protein. PGs have signaling roles and modulatory functions in the extracellular matrix and at the cell surface. To elucidate the functions of higher-order PG structures, pseudoPGs that imitate the PG structure were prepared to develop probes and affinity adsorbents. Poly-L: -lysine (PLL) or polyacrylamide (PAA) was coupled with various GAGs, then biotinylated, and the remaining amino groups were blocked to obtain the pseudoPG probes, biotinyl PLL (BPL)- or PAA (BPA)-GAGs. Lactoferrin exhibited 30-times higher affinity toward BPL-heparin than the conventional single-strand probe, biotin-hydrazide-heparin. Heparin-PLL was immobilized on a formyl-Sepharose and compared with the Hep-Sepharose in which heparin was directly immobilized to amino-Sepharose. Screening for ligands in normal rat brain revealed several proteins that specifically bound to either of the two adsorbents, indicating that the heparin-binding proteins exhibit specific recognition depending on the higher-order structure of the PG.

Autoactivation of pancreatic trypsinogen is controlled by carbohydrate-specific interaction.

Activation of bovine pancreatic trypsinogen (BPTG) by trypsin (BPT) was found to be inhibited by d GalN/GalNAc at pH 5.5, the pH of secretory granules in the pancreas. Binding studies with biotinylated sugar-polymers indicated that BPTG and BPT bind to α-GalNAc, α-Man, and α-Gal better at pH 5.5 than at pH 7.5. Ultraviolet-difference spectra indicated that BPTG binding to α-GalNAc differs substantially from BPTG binding to other sugars. The N-α-benzoyl-d,l-arginine-p-nitroanilide hydrochloride-hydrolyzing activity of BPT was only slightly affected by these sugars. The results indicate that the binding of GalNAc - containing glycoconjugates protects BPTG from autoactivation, and this may be a self-defense mechanism against intrapancreatic activation.

Immobilized glycosylated Fmoc-amino acid for SPR: comparative studies of lectin-binding to linear or biantennary diLacNAc structures.

A method to immobilize glycan-linked amino acids with protected α-amino groups, which are key intermediates to produce the desired neoglycoprotein, to a Biacore sensor chip was developed and its utility for interaction analyses was demonstrated. Two types of diN-acetyllactosamine (diLacNAc)-containing glycans, a core 2 hexasaccharide involving linear diLacNAc that is O-linked to N-(9-fluorenyl)methoxycarbonyl (Fmoc)-Thr and a biantennary diLacNAc that is N-linked to Fmoc-Asn, were used as ligands. For immobilization, the free carboxyl groups of the amino acid residues were activated with EDC/NHS, then reacted with the ethylenediamine-derivatized carboxymethyldextran sensor chip to obtain the desired ligand concentrations. Interactions of the ligands with five plant lectins were analyzed by surface plasmon resonance, and the bindings were compared. The resonance unit of each lectin was corrected by subtracting that of the reference cell on which the Fmoc-Thr-core 1 or Fmoc-Asn was immobilized as a ligand. The carbohydrate specificities of interactions were verified by preincubating lectins with their respective inhibitory sugar before injection. By steady state analysis, the Lycopersicon esculentum lectin showed a 27-fold higher affinity to linear diLacNAc than to biantennary diLacNAc, while Datura stramonium and Solanum tuberosum lectins both showed low Ka,apps of 10(6)M(-1) for these two ligands. In contrast, Ricinus communis agglutinin-120 showed a 3.2-fold higher Ka,app to biantennary LacNAc than to linear diLacNAc. A lectin purified from Pleurocybella porrigens mushroom interacted at the high affinity of 10(8)M(-1) with both linear and biantennary diLacNAcs, which identified it as a unique probe. This method provides a useful and sensitive system to analyze interactions by simulating the glycans on the cell surface.

Novel anti-HIV-1 activity produced by conjugating unsulfated dextran with polyL-lysine.

A conjugate of polyL-lysine (PLL) with unsulfated dextran produced by reductive amination was found to have remarkable anti-HIV-1 activity against both the macrophage-tropic R5 virus Ba-L and T-cell line tropic X4 virus IIIB strains, although neither PLL nor dextran has such activity. The conjugate is a pseudoproteoglycan (pseudoPG) that simulates the structure of a proteoglycan. Conjugation with dextran was found to produce an antiviral effect in three kinds of assay systems including a human CD4(+) T-cell line, and the pseudoPG synthesized using 10 kDa PLL and 10 kDa dextran showed EC(50) 4-40 times lower than that of sulfated dextran or heparin against Ba-L and EC(50) equal to that against IIIB, indicating that PLL-dextran (PLL-Dex) was more effective against R5 virus than sulfated polysaccharides. PLL-Dex significantly suppressed a clinically isolated R5 virus from primary peripheral blood mononuclear cells. PLL-Dex interacted with the virus during adsorption to the cell and also decreased virus entry into the cell, suggesting PLL-Dex has multiple preventive mechanisms against HIV-1.

Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences.

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.