Role of glycosphingolipid-enriched microdomains in innate immunity: microdomain-dependent phagocytic cell functions

The innate immune system is the first line of defense against pathogenic microorganisms, such as bacteria, fungi, and viruses. Phagocytes, such as neutrophils and macrophages, play an important role in the innate immune system by recognizing, engulfing, and eliminating pathogens. It has been suggested that lipid membrane microdomains/rafts of phagocytes are involved in these innate immune responses, including superoxide generation, cell migration, and phagocytosis. Lactosylceramide (LacCer), a neutral glycosphingolipid, forms glycosphingolipid-enriched microdomains together with the Src family kinase, Lyn, on the neutrophil plasma membrane. LacCer-enriched microdomains have been suggested to play important roles in innate immune function of neutrophils. However, the molecular mechanisms underlying these phenomena remain largely unknown. Recent proteomic analyses of microdomains from phagocytes have provided insight into membrane microdomain-mediated functions in the processes of phagocytosis. In this review, we discuss the membrane microdomain-associated immune functions of phagocytes, focusing on those functions of LacCer-enriched microdomains and recent proteomic approaches to determine the molecular mechanisms underlying these functions.     

Accurate determination of leucine and valine side-chain conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] isotope labeling, NOE pattern recognition, and methine Cgamma-Hgamma/Cbeta-Hbeta residual dipolar couplings: application to the 34-kDa enzyme IIA(chitobiose)

An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a ¹⁵N/¹³C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)–H_N(i) and Hγ(i)–H_N(i+1) NOEs in a 3D ¹⁵N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine ¹³C–¹H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIA^Chitobiose, a 34 kDa homotrimeric phosphotransferase protein.     

Role of CD44 in epithelial wound repair: migration of rat hepatic stellate cells utilizes hyaluronic acid and CD44v6

The hyaluronic acid receptor, CD44, exists as multiple splice variants that appear to have a role in migration of tumor cells. The role of this receptor and its variants in normal wound repair is poorly understood. A central feature of wound repair in the liver is activation and migration of perisinusoidal stellate cells. We have examined CD44 expression by stellate cells from normal or injured rat liver, finding that it increases with injury and involves a distinct set of CD44 splice variants. Among the latter, variants containing the v6 exon (CD44v6) are strikingly increased. Analysis of migration of primary cells on transwell filter inserts reveals that only cells isolated from injured liver are migratory. Also, they move more rapidly on hyaluronic acid than on collagen I or collagen IV. A polyclonal antibody to recombinant CD44v6 blocks migration by 50%, whereas antibody to CD44v4 has no effect. The inhibition is specific for cells migrating on hyaluronic acid and is reversed by synthetic peptide representing the N terminus of the v6 protein. In conclusion, activated stellate cells use CD44v6 and hyaluronic acid for migration. Given the evidence that migration is required for progression of injury with scar formation, blockers of CD44v6 expression or function are candidates for preventing the deleterious effects of chronic fibrosis.     

Structure-based design of P3 moieties in the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. Structure-based designs of the P3 moiety in the peptide mimetic factor VIIa inhibitor successfully lead to novel inhibitors with selectivity for FVIIa/TF and extrinsic coagulation the same as or even higher than those of previously reported peptide mimetic factor VIIa inhibitors. X-ray crystal structure analysis reveals that one of the novel inhibitors shows improved selectivity by forming interactions between the inhibitor and FVIIa as expected. Another of the novel inhibitors achieves improved selectivity through an unexpected hydrogen bond with Gln217, with a unique bent conformation in FVIIa/TF accompanied by conformational changes of the inhibitor and the protein.     

Crk associates with ERM proteins and promotes cell motility toward hyaluronic acid

Cell migration is a well organized process regulated by the extracellular matrix-mediated cytoskeletal reorganization. The signaling adaptor protein Crk has been shown to regulate cell motility, but its precise role is still under investigation. Herein, we report that Crk associates with ERM family proteins (including ezrin, radixin, and moesin), activates RhoA, and promotes cell motility toward hyaluronic acid. The binding of Crk with ERMs was demonstrated both by transient and stable protein expression systems in 293T cells and 3Y1 cells, and it was shown that v-Crk translocated the phosphorylated form of ERMs to microvilli in 3Y1 cells by immunofluorescence and immunoelectron microscopy. This v-Crk-dependent formation of microvilli was suppressed by inhibitors of Rho-associated kinase, and the activity of RhoA was elevated by coexpression of c-Crk-II and ERMs in 3Y1 cells. In concert with the activation of RhoA by Crk, Crk was found to associate with Rho-GDI, which has been shown to bind to ERMs. Furthermore, upon hyaluronic acid treatment, coexpression of c-Crk-II and ERMs enhanced cell motility, whereas the sole expression of c-Crk-II or either of the ERMs decreased the motility of 3Y1 cells. These results suggest that Crk may be involved in regulation of cell motility by a hyaluronic acid-dependent mechanism through an association with ERMs.     

Induction and Purification of Endo-β-N-Acetylglucosaminidase from Arthrobacter protophormiae Grown in Ovalbumin

Arthrobacter protophormiae produced a high level of extracellular endo-β-N-acetylglucosaminidase when cells were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction of ovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine, and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the range of pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysis rates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormiae endo-β-N-acetylglucosaminidase was very similar to that of Endo-C_II from Clostridium perfringens. Therefore, the enzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme.     

Enzymatic synthesis of novel oligosaccharides by use of transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

The transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae was used for the enzymatic synthesis of novel oligosaccharides. When (Man)6(GlcNAc)2Asn was used as a substrate for the transglycosylation, (Man)6GlcNAc-Glc, (Man)6GlcNAc-Man, (Man)6GlcNAc-chitobiose, and (Man)6GlcNAc-gentiobiose were synthesized. Their structures were identified by HPLC, ion spray mass spectrometry, and digestion with glycosidases. Endo-beta-N-acetylglucosaminidases hydrolyzed the pyridylamino derivatives of these oligosaccharides.     

Studies on Biosynthesis of Biotin by Microorganisms

A biotinless mutant (K-681-UV-134) accumulated a large amount of desthiobiotin and an unknown biotin-vitamer in the culture medium.

The parent strain (K-681) of this mutant isolated from soil was identified as Bacillus cereus.

The unknown vitamer was accumulated at the early stage of the incubation in comparison with desthiobiotin.

The unknown vitamer was purified by the paper- and column-chromatographic methods from the culture filtrate. The purified vitamer gave a single spot when spraying with the ninhydrin reagent after paper chromatographing and its R_F values in several solvent systems were identical with those of authentic 7-keto-8-aminopelargonic acid.