A new PEGylation reagent enabling selective modification of free thiol groups is described in this article. The reagent was synthesized by attaching linear polyethylene glycol (PEG) N-hydroxysuccinimide to selenocystamine. The reaction was very fast, resulting in over 95% conversion yield. The active group of this new PEG-Se reagent is a diselenide, reacting with thiols via thiol/diselenide exchange reaction. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) with an unpaired cysteine at the position 18 (Cys18) was used as a model protein. It was comparatively PEGylated with the new PEG-Se reagent, as well as with commercially available maleimide (PEG-Mal) and ortho-pyridyl disulfide (PEG-OPSS) PEG reagents. The highest PEGylation yield was obtained with PEG-Mal, followed by PEG-OPSS and PEG-Se. The reaction rates of PEG-Mal and PEG-Se were comparable, while the reaction rate of PEG-OPSS was lower. Purified monoPEGylated rhG-CSF conjugates were characterized and compared. Differences in activity, stability, and in vivo performance were observed, although all conjugates contained a 20 kDa PEG attached to the Cys18. Minor conformational changes were observed in the conjugate prepared with PEG-Mal. These changes were also reflected in low in vitro biological activity and aggregate formation of the maleimide conjugate. The conjugate prepared with PEG-Se had the highest in vitro biological activity, while the conjugate prepared with PEG-OPSS had the best in vivo performance.
Cytochrome P450 lanosterol 14alpha-demethylase (CYP51) and its products meiosis-activating sterols (MAS) were hypothesized by previous in vitro studies to have an important role in regulating meiosis and reproduction. To test this in vivo we generated a conditional male germ cell-specific knockout of the gene Cyp51 in the mouse. High excision efficiency of Cyp51 allele in germ cells resulted in 85-89% downregulation of Cyp51 mRNA and protein levels in germ cells. Quantitative metabolic profiling revealed significantly higher levels of CYP51 substrates lanosterol and 24,25-dihydrolanosterol and substantially diminished levels of MAS, the immediate products of CYP51. However, germ cell-specific ablation of Cyp51, leading to lack of MAS, did not affect testicular morphology, daily sperm production or reproductive performance in males. It is plausible that due to the similar structures of cholesterol intermediates, previously proposed biological function of MAS in meiosis progression can be replaced by some other yet unidentified functionally redundant lipid molecule(s). Our results using the germ cell-specific knockout model provides first in vivo evidence that the de novo synthesis of MAS and cholesterol in male germ cells is most likely not essential for spermatogenesis and reproduction and that MAS, originating from germ cells do not cell-autonomously regulate spermatogenesis and fertility.
330a Contraction stimulates Na+,K+-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na+,K+-ATPase activity in skeletal muscle remains to be determined. Short-term stimulation of rat L6 myotubes with the AMPK activator AICAR, activates AMPK and promotes translocation of the Na+,K+-ATPase alpha1-subunit to the plasma membrane and increases Na+,K+-ATPase activity as assessed by ouabain-sensitive 86Rb+-uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increases AMPK phosphorylation and Na+,K+-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na+,K+-ATPase activity. The effect of AICAR on Na+,K+-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na+,K+-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK alpha-subunits. AMPK stimulation leads to Na+,K+-ATPase alpha1-subunit dephosphorylation at Ser18, which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na+,K+-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A/PME-1 complex in AMPK-mediated regulation of Na+,K+-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.
Background: A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs - complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes. Methods: An original algorithmic approach (Global Sequence Signature-GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences. Results: Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation. Conclusions: Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop. General significance: In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.
We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. Biological Significance: DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes.