The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.
Cell-surface carbohydrates mediate the adherence of many pathogenic bacteria to epithelial cells. Because gram-negative bacteria adhere especially well to respiratory epithelial cells obtained from severely ill patients, we compared respiratory epithelial cell-surface carbohydrate levels of normal subjects with those of critically ill patients. Lectins were used to quantitate the amount of mannose, galactose, fucose, and sialic acid on buccal and tracheal cells. Fifteen critically ill patients, 20 normal subjects, and 10 minimally ill hospitalized patients were studied. The severely ill patients' buccal cells had decreased amounts of sialic acid and galactose. No differences were found between the normal and critically ill patients' tracheal-cell carbohydrates. The results obtained with a sialic acid-specific lectin were confirmed by direct measurement of buccal-cell sialic acid. We conclude that severely ill patients have decreased amounts of galactose and sialic acid on their upper-airway epithelial cells, and that loss of these two monosaccharides may explain the high prevalence of gram-negative bacterial colonization and pneumonia in the critically ill.
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