Deciphering the complex three-way interaction between the non-integrin laminin receptor, galectin-3 and Neisseria meningitidis

The non-integrin laminin receptor (LAMR1/RPSA) and galectin-3 (Gal-3) are multi-functional host molecules with roles in diverse pathological processes, particularly of infectious or oncogenic origins. Using bimolecular fluorescence complementation and confocal imaging, we demonstrate that the two proteins homo- and heterodimerize, and that each isotype forms a distinct cell surface population. We present evidence that the 37 kDa form of LAMR1 (37LRP) is the precursor of the previously described 67 kDa laminin receptor (67LR), whereas the heterodimer represents an entity that is distinct from this molecule. Site-directed mutagenesis confirmed that the single cysteine (C173) of Gal-3 or lysine (K166) of LAMR1 are critical for heterodimerization. Recombinant Gal-3, expressed in normally Gal-3-deficient N2a cells, dimerized with endogenous LAMR1 and led to a significantly increased number of internalized bacteria (Neisseria meningitidis), confirming the role of Gal-3 in bacterial invasion. Contact-dependent cross-linking determined that, in common with LAMR1, Gal-3 binds the meningococcal secretin PilQ, in addition to the major pilin PilE. This study adds significant new mechanistic insights into the bacterial–host cell interaction by clarifying the nature, role and bacterial ligands of LAMR1 and Gal-3 isotypes during colonization

Studies of reactions within molecular complexes: alkaline hydrolysis of substituted phenyl benzoates in the presence of xanthines

Complexation with caffeine and theophylline-7-acetate depresses the rate of alkaline hydrolysis of substituted phenyl benzoates and is consistent with the formation of molecular complexes with 1 : 1 stoichiometry between the hosts and esters; stacking of the xanthines is excluded as an explanation in the range of concentrations studied. Bronsted-type correlations have been determined for the rate and complexation constants and for the transition-state binding constants, Development of effective charge in the transition state of the reactions in bulk solvent is slightly less than that in the host-ester complex, consistent with a similar electronic environment in both states, The negative Bronsted beta values for K-s indicate that the interactions between ester and hosts involve electron donation to the host from the ester, Inhibition of hydrolysis is attributed to repulsion of the hydroxide ion from the host-ester complex by the extra hydrophobicity engendered by the xanthine host, as well as by the weaker binding of the transition state to the host compared with that in the host-ester complex

Enhanced alkaline hydrolysis of monoesterified 4-tert-butylcalix[4]arenes involving intramolecular electrophilic catalysis by the phenolic hydroxy group

Rate enhancements over model systems up to 1600-fold are observed in the alkaline hydrolysis of monobenzoate esters of calix[4]arenes. Spectrophotometric titration of the mono-benzoate ester over a pH range indicates ionisation of phenolic hydroxy groups at pK 6.84,12.14 and > 14.02. The kinetics of hydrolysis of substituted monobenzoate esters of 4-tert-butylcalix[4]arene in 50% (v/v) ethanol-water solvent (at 25-degrees-C) obey pseudo-first-order kinetics which fit the rate law, k(obs) = (k1K(w)/K(a)' + k2[OH])[OH/(K(w)/K(a)' + [OH]) where k, and k2 correspond to bimolecular attack of hydroxide ion on monoanion and dianion respectively. The kinetics were measured at pHs at which the calixarene esters are in their monoanionic form. The kinetics of the alkaline hydrolyses (k(OH)) of substituted benzoate esters of 4-nitrophenol were measured under the same conditions. The following Hammett equations are obeyed. log k1 = 1.86sigma + 1.33 log k2 = 2.21sigma + 0.34 pK(a)' = -2.90sigma + 11.78 log k(OH) = 2.23sigma + 0.26 The large negative Hammett rho value for the pK(a)' of the calixarenes (determined kinetically) is consistent with a strong interaction between the ester and the ionised hydroxy groups, attributed to formation of an intramolecular tetrahedral adduct. The formation of the adduct means that hydrolysis is retarded and the enhancements observed are lower limits. The alkaline hydrolysis of the calixarene esters is due to hydroxide ion attack on monoanion for k1 and on the dianion for k2. The substantial negative Hammett rho values for water attack on dianion and trianion respectively provide unequivocal evidence to exclude these mechanisms in favour of hydroxide ion attack

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr(268); previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr(268) led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(beta 1-3)-GalNAc(beta 1-4)-GalNAc(beta 1-4)-GalNAca1-Thr(268); modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr(268) promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis