Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity

Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application

Insights into the CRISPR/Cas system of <it>Gardnerella vaginalis</it>

<p>Abstract</p> <p>Background</p> <p><it>Gardnerella vaginalis</it> is identified as the predominant colonist of the vaginal tracts of women diagnosed with bacterial vaginosis (BV). <it>G. vaginalis</it> can be isolated from healthy women, and an asymptomatic BV state is also recognised. The association of <it>G. vaginalis</it> with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparate gene content. The contribution of horizontal gene transfer to shaping the genomes of <it>G. vaginalis</it> is acknowledged. The CRISPR loci of the recently discovered CRISPR/Cas microbial defence system provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements.</p> <p>Results</p> <p>The CRISPR/Cas loci were analysed using available sequence data from three <it>G. vaginalis</it> complete genomes and 18 <it>G. vaginalis</it> draft genomes in the NCBI database, as well as PCR amplicons of the genomic DNA of 17 clinical isolates. The <it>cas</it> genes in the CRISPR/Cas loci of <it>G. vaginalis</it> belong to the E. coli subtype. Approximately 20% of the spacers had matches in the GenBank database. Sequence analysis of the CRISPR arrays revealed that nearly half of the spacers matched <it>G. vaginalis</it> chromosomal sequences. The spacers that matched <it>G. vaginalis</it> chromosomal sequences were determined to not be self-targeting and were presumably neither constituents of mobile-element-associated genes nor derived from plasmids/viruses. The protospacers targeted by these spacers displayed conserved protospacer-adjacent motifs.</p> <p>Conclusions</p> <p>The CRISPR/Cas system has been identified in about one half of the analysed <it>G. vaginalis</it> strains. Our analysis of CRISPR sequences did not reveal a potential link between their presence and the virulence of the <it>G. vaginalis</it> strains. Based on the origins of the spacers found in the <it>G. vaginalis</it> CRISPR arrays, we hypothesise that the transfer of genetic material among <it>G. vaginalis</it> strains could be regulated by the CRISPR/Cas mechanism. The present study is the first attempt to determine and analyse the CRISPR loci of bacteria isolated from the human vaginal tract.</p

Monoclonal antibodies raised against 167–180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity

Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application