Epidermal-specific deletion of CD44 reveals a function in keratinocytes in response to mechanical stress

CD44, a large family of transmembrane glycoproteins, plays decisive roles in physiological and pathological conditions. CD44 isoforms are involved in several signaling pathways essential for life such as growth factor-induced signaling by EGF, HGF or VEGF. CD44 is also the main hyaluronan (HA) receptor and as such is involved in HA-dependent processes. To allow a genetic dissection of CD44 functions in homeostasis and disease, we generated a Cd44 floxed allele allowing tissue- and time-specific inactivation of all CD44 isoforms in vivo. As a proof of principle, we inactivated Cd44 in the skin epidermis using the K14Cre allele. Although the skin of such Cd44Δker mutants appeared morphologically normal, epidermal stiffness was reduced, wound healing delayed and TPA induced epidermal thickening decreased. These phenotypes might be caused by cell autonomous defects in differentiation and HA production as well as impaired adhesion and migration on HA by Cd44Δker keratinocytes. These findings support the usefulness of the conditional Cd44 allele in unraveling essential physiological and pathological functions of CD44 isoforms.We are grateful to Professor Ben Wielockx (DIPP,Dresden, Germany) for help with the in vivo wound healing assay, to Professors Pierre Chambon and Daniel Metzger (IGBMC, Strasbourg, France) for the kind gift of the K14 Cre mice. We thank Karin Müller-Decker (DKFZ, Heidelberg, Germany) and Peter Angel (DKFZ) for their help with the TPA induction experiments. We are extremely grateful to the animal facility of our institute (ITG, KIT) and especially to Selma Huber for their help with animal experiments. We also want to thank R. Saffrich. (Department of Medicine V (Hematology, Oncology & Rheumatology, University of Heidelberg, Heidelberg) for technical assistance in time-lapse imaging. We are grateful to Julia Gutjahr (Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria) for her help with the immunohistological pictures. We also thank Ana Guío-Carrión (Spanish National Cancer Centre, Genes Development and Disease Group, Cancer Cell Biology Programme, Madrid, Spain) for technical assistance. MT and ASB thank the German Research Foundation (Collaborative Research Center, CRC 873 B07) for support. ASB thank C. Monzel (Laboratoire Physico-Chimie, Institut Curie, Paris, France) for assistance in data analysis. iCeMS is supported by World Premier International Research Center Initiative (WPI), MEXT (Japan). EFW and LB are supported by grants from the Spanish Ministry of Economy (BFU2012 – 40230, and SAF2015 – 70857, co-funded by the ERDF-EU)S

Improvement of a fermentation process for the production of two PfAMA1-DiCo-based malaria vaccine candidates in Pichia pastoris

Pichia pastoris is a simple and powerful expression platform that has the ability to produce a wide variety of recombinant proteins, ranging from simple peptides to complex membrane proteins. A well-established fermentation strategy is available comprising three main phases: a batch phase, followed by a glycerol fed-batch phase that increases cell density, and finally an induction phase for product expression using methanol as the inducer. We previously used this three-phase strategy at the 15-L scale to express three different AMA1-DiCo-based malaria vaccine candidates to develop a vaccine cocktail. For two candidates, we switched to a two-phase strategy lacking the intermediate glycerol fed-batch phase. The new strategy not only provided a more convenient process flow but also achieved 1.5-fold and 2.5-fold higher space-time yields for the two candidates, respectively, and simultaneously reduced the final cell mass by a factor of 1.3, thus simplifying solid-liquid separation. This strategy also reduced the quantity of host cell proteins that remained to be separated from the two vaccine candidates (by 34% and 13%, respectively), thus reducing the effort required in the subsequent purification steps. Taken together, our new fermentation strategy increased the overall fermentation performance for the production of two different AMA1-DiCo-based vaccine candidates

Production, quality control, stability and pharmacotoxicity of a malaria vaccine comprising three highly similar PfAMA1 protein molecules to overcome antigenic variation

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region be

Hepatocyte Growth Factor-induced Ras Activation Requires ERM Proteins Linked to Both CD44v6 and F-Actin

In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a “signalosome” complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a “responder” to signal transduction. Here, we show now that F-actin acts as an “inducer” that actively organizes the signaling cascade