Recombinant expression of hydroxylated human collagen in Escherichia coli

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26% were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications

Congenital disorders of glycosylation: a concise chart of glycocalyx dysfunction

Glycosylation is a ubiquitous modification of lipids and proteins. Despite the essential contribution of glycoconjugates to the viability of all living organisms, diseases of glycosylation in humans have only been identified over the past few decades. The recent development of next-generation DNA sequencing techniques has accelerated the pace of discovery of novel glycosylation defects. The description of multiple mutations across glycosylation pathways not only revealed tremendous diversity in functional impairments, but also pointed to phenotypic similarities, emphasizing the interconnected flow of substrates underlying glycan assembly. The current list of 100 known glycosylation disorders provides an overview of the significance of glycosylation in human development and physiology

A comprehensive multi-analyte method for hair analysis: Substance-specific quantification ranges and tool for task-oriented data evaluation

The aim of the present study was to quantify a large number of analytes including opioids, stimulants, benzodiazepines, z-drugs, antidepressants and neuroleptics within a single sample workup followed by a single analytical measurement. Expected drug concentrations in hair are strongly substance dependent. Therefore, three different calibration ranges were implemented: 0.5 to 600 pg/mg (group 1), 10 to 12,000 pg/mg (group 2) and 50 to 60,000 pg/mg (group 3). In order to avoid saturation effects, different strategies were applied for selected transitions including the use of parent mass ions containing one or two 13C-isotopes and detuning of the declustering potential and/or collision energy. Drugs were extracted from pulverized hair by a two-step extraction protocol and measured by liquid chromatrography-tandem mass spectrometry (LC-MS-MS) using Scheduled MRM™ Algorithm Pro. In total, 275 MRM transitions including 43 deuterated standards were measured. The method has been fully validated according to international guidelines. A MultiQuant™ software based tool for task-oriented data evaluation was established, which allows extracting selected information from the measured data sets. The matrix effects and recoveries were within the allowed ranges for the majority of the analytes. The lower limits of quantification (LLOQs) were for ∼72% of the analytes in the low-pg/mg range (0.5–5 pg/mg) and for ∼24% of the analytes between 10 and 50 pg/mg. These LLOQs considered cut-offs by the Society of Hair Testing (SoHT), if recommended. The herein established multi-analyte approach meets the specific requirements of forensic hair testing and can be used for the rapid and robust measurement of a wide range of psychoactive substances. The analyte-specific wide concentration ranges open up a wide field of applications

Recombinant expression of hydroxylated human collagen in Escherichia coli

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications

T cells dampen innate immune responses through inhibition of NLRP1 and NLRP3 inflammasomes.

Inflammation is a protective attempt by the host to remove injurious stimuli and initiate the tissue healing process. The inflammatory response must be actively terminated, however, because failure to do so can result in 'bystander' damage to tissues and diseases such as arthritis or type-2 diabetes. Yet the mechanisms controlling excessive inflammatory responses are still poorly understood. Here we show that mouse effector and memory CD4(+) T cells abolish macrophage inflammasome-mediated caspase-1 activation and subsequent interleukin 1beta release in a cognate manner. Inflammasome inhibition is observed for all tested NLRP1 (commonly called NALP1) and NLRP3 (NALP3 or cryopyrin) activators, whereas NLRC4 (IPAF) inflammasome function and release of other inflammatory mediators such as CXCL2, interleukin 6 and tumour necrosis factor are not affected. Suppression of the NLRP3 inflammasome requires cell-to-cell contact and can be mimicked by macrophage stimulation with selected ligands of the tumour necrosis factor family, such as CD40L (also known as CD40LG). In a NLRP3-dependent peritonitis model, effector CD4(+) T cells are responsible for decreasing neutrophil recruitment in an antigen-dependent manner. Our findings reveal an unexpected mechanism of inflammasome inhibition, whereby effector and memory T cells suppress potentially damaging inflammation, yet leave the primary inflammatory response, crucial for the onset of immunity, intact