Laura Hirvas

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLex and sLea respectively) decorated ligands. Endothelial cells have been shown to express sLex epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLex on sialylated N-acetyllactosamine via the action of α(1,3)fucosyltransferase(s), endothelial cells can also degrade sLex to Lewis x through the action of α(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLex, which facilitates their adhesion to endothelial E- and P-selectin.

The Ssc protein of enteric bacteria has significant homology to the acyltransferase Lpxa of lipid A biosynthesis, and to three acetyltransferases

The Ssc protein, a novel essential protein affecting the function of the enterobacterial outer membrane, matched in a protein homology search best with LpxA (UDP‐N‐acetylglucosamine 3‐hydroxymyristoyl transferase), the enzyme which catalyzes the first step of lipid A biosynthesis. The corresponding genes, located O.56 kb apart, were 46.7% identical. The search also revealed homology to the bacterial acetyltransferases LacA and NodL, as well as to a hypothetical protein Yglm. The region of residues 109–149 Ssc displayed the highest homology and was also homologous with another bacterial acetyltransferase, CysE, and three other bacterial proteins, two of which are hypothetical. This region and the corresponding regions of all other proteins were found to have a peculiar repeated hexapeptide pattern. Each hexapeptide unit starts with isoleucine (or its equivalent leucine and valine). In most units, the second residue is glycine and the fifth residue either valine or alanine.

Complete sequence of the ompH gene encoding the 16-kDa cationic outer membrane protein of Salmonella typhimurium.

The complete nucleotide sequence of the ompH gene encoding the 16-kDa basic outer membrane protein of Salmonella typhimurium was determined. The OmpH protein is synthesized in a precursor form with additional 20 amino acid residues in the N terminus of the protein. This peptide has common characteristics of signal sequences. The promoter region has strong homology to consensus sequences of Escherichia coli. The expression of ompH was detected in minicells.

Bacterial 'histone-like protein I' (HLP-I) is an outer membrane constituent?

The nucleoid‐associated ‘histone‐like protein I’ (HLP‐I) protein of E. coli was found to be homologous with the cationic 16‐kDa outer membrane protein OmpH of Salmonella typhimurium. Deduced from the nucleotide sequence, the HLP‐I protein has 91% identical residues with the OmpH protein. Both proteins have very similar cleavable signal sequences. The nucleotide sequence similarity between the corresponding genes hlpA and ompH is 87%. The ompH gene is located in a gene cluster resembling the hlpA‐ORF17 region of E. coli which is close to the Ipx genes involved in the biosynthesis of lipopolysaccharides. The localization of the OmpH/HLP‐I protein in the cell is discussed.

The Centrally Acting β1,6N-Acetylglucosaminyltransferase (GlcNAc to Gal) FUNCTIONAL EXPRESSION, PURIFICATION, AND ACCEPTOR SPECIFICITY OF A HUMAN ENZYME INVOLVED IN MIDCHAIN BRANCHING OF LINEAR POLY-N-ACETYLLACTOSAMINES

In the present experiments the cDNA coding for a truncated form of the β1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67–74-kDa fusion protein. Typically a yield of 750 μg of the purified protein/liter of suspension culture was obtained. The purified recombinant protein catalyzed the transfer of GlcNAc from UDP-GlcNAc to the linear tetrasaccharide Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc, converting the acceptor to the branched pentasaccharide Galβ1–4GlcNAcβ1–3(GlcNAcβ1–6)Galβ1–4GlcNAc as shown by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, degradative experiments, and 1H NMR spectroscopy of the product. By contrast, the recombinant enzyme did not catalyze any reaction when incubated with UDP-GlcNAc and the trisaccharide GlcNAcβ1–3Galβ1–4GlcNAc. Accordingly, we call the recombinant β1,6-GlcNAc transferase cIGnT6 to emphasize its action atcentral rather than peridistal galactose residues of linear polylactosamines in the biosynthesis of blood group I antigens. Taken together this in vitro expression of I-branching enzyme, in combination with the previously cloned enzymes, β1,4galactosyltransferase and β1,3N-acetylglucosaminyltransferase, should allow the general synthesis of polylactosamines based totally on the use of recombinant enzymes.

The outer membrane of lipid A-deficient Escherichia coli mutant LH530 has reduced levels of OmpF and leaks periplasmic enzymes

We have previously described a new Escherichia coli K-12 mutant, LH530, which has a defective outer membrane. LH530 is very sensitive to hydrophobic antibiotics, does not grow at 42 degrees C and synthesizes reduced amounts of lipid A. Phenotypically LH530 is very similar to the known lipid A biosynthesis mutants of E. coli and Salmonella typhimurium. Its genetic defect is not known, but the defect is suppressed by multiple copies of ORF195. Here we show that at 37 degrees C LH530 contains a reduced amount of the OmpF porin and that it leaks periplasmic beta-lactamase at 37 degrees C and 42 degrees C. We further show that ORF195, when present at low copy number, restores the antibiotic resistance and lipid A biosynthesis of LH530 at 28 degrees C, but not at higher temperatures. In contrast, OmpF expression is restored at 37 degrees C.

Primary structure and expression of the Ssc-protein of Salmonella typhimurium.

A 1020-bp open reading frame (ORF) was found immediately downstream of the ompH gene of Salmonella typhimurium. This ORF (ORF-36) encodes a moderately hydrophobic protein with 341 amino acid residues (calculated molecular mass, 35,928 Da). The ORF-36 product was detected in minicells. Downstream of ORF-36, another ORF was found. It is highly homologous to the E. coli ORF (ORF-17.4) which precedes the lpx-genes involved in lipid A biosynthesis. ORF-36 is probably analogous to the firA gene of E. coli, the sequence of which has not yet been published. Thus it appears that the enterobacterial ompH and lpx genes are separated only by the ORF-36 and ORF-17.4 genes. We also discuss the data on the function of the ORF-36 protein. On this basis, we suggest that the protein could be called the Ssc protein.

The Lipid a Biosynthesis Deficiency of the Escherichia Coli Antibiotic-Supersensitive Mutant LH530 is Suppressed by a Novel Locus, ORF195

A new mutant of Escherichia coli K-12 supersensitive to both hydrophobic and large hydrophilic antibiotics was isolated and characterized. The mutant grew well at 28 degrees C, poorly at 37 degrees C, and did not grow at 42 degrees C. The rate of its lipid A biosynthesis was reduced as compared to that of the parent strain. This deficiency was rescued by a novel locus, ORF195, the function of which has not been elucidated. ORF195 is located in the 76 min region in the E. coli chromosome and encodes a hypothetical 21.8 kDa protein with no signal sequence. ORF195 isolated from the mutant strain had an identical sequence to the wild-type allele, indicating a suppressor function of the gene product.

The nucleotide and deduced amino acid sequence of the cationic 19 kDa outer membrane protein OmpH of Yersinia pseudotuberculosis

The OmpH proteins of enteric bacteria are recently described, small (16 kDa), cationic outer membrane proteins. Because a Yersinia pseudotuberculosis cell envelope protein of this size has been found to cross-react serologically with the human histocompatibility antigen HLA-B27 (B*2701), the sequence of Y. pseudotuberculosis OmpH was determined by sequencing the gene region which encodes mature OmpH. A protein consisting of 143 amino acid residues was found. It was 96% homologous with the OmpH of Y. enterocolitica and 62% homologous with that of Escherichia coli. Two separate OmpH regions had sequence similarity with B*2701; they were identical in both Yersinia species.