Limulus Clotting Factor C is a multi-domain serine protease that triggers horseshoe crab hemolymph clotting in the presence of trace amounts of bacterial lipopolysaccharides. Here we describe and functionally characterize an homologous molecule, designated as IrFC, from the hard tick Ixodes ricinus. Tick Factor C consists of an N-terminal cysteine-rich domain, four complement control protein (sushi) modules, an LCCL domain, a truncated C-lectin domain and a C-terminal trypsin-type domain. Developmental expression profiling by quantitative real-time PCR revealed that the irfc mRNA is expressed in all stages including eggs. In tissues dissected from adult I. ricinus females, the irfc mRNA is present mainly in tick hemocytes and accordingly, indirect immunofluorescence microscopy localized IrFC intracellularly, in tick hemocytes. Irfc mRNA levels were markedly increased upon injection of sterile saline, or different microbes, demonstrating that the irfc gene transcription occurs in response to injury. This indicates a possible role of IrFC in hemolymph clotting and/or wound healing, although these defense mechanisms have not been yet definitely demonstrated in ticks. RNAi silencing of irfc expression resulted in a significant reduction in phagocytic activity of tick hemocytes against the Gram-negative bacteria Chryseobacterium indologenes and Escherichia coli, but not against the yeast, Candida albicans. This result suggests that IrFC plays a role in the tick primordial complement system and as such possibly mediates transmission of tick-borne pathogens.

• MeSH
• Borrelia imunologie   MeSH
• Candida albicans imunologie   MeSH
• Escherichia coli imunologie   MeSH
• exprese genu MeSH
• fagocytóza MeSH
• klíště enzymologie   genetika   imunologie   mikrobiologie   MeSH
• komplement fyziologie   MeSH
• messenger RNA biosyntéza   genetika   MeSH
• Micrococcus luteus imunologie   MeSH
• molekulární sekvence - údaje MeSH
• prekurzory enzymů biosyntéza   genetika   MeSH
• přirozená imunita MeSH
• proteiny členovců biosyntéza   genetika   MeSH
• serinové endopeptidasy biosyntéza   genetika   MeSH
• upregulace imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.

• MeSH
• beta-N-acetylhexosaminidasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• fungální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• glykosylace MeSH
• katalytická doména MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• konzervovaná sekvence MeSH
• mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• Penicillium enzymologie   genetika   MeSH
• prekurzory enzymů chemie   genetika   izolace a purifikace   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH