Increased trans-glycosylation activity of hexosaminidases for synthesis of human milk oligosaccharides

It is well known that the composition of human breast milk differs significantly from the one of ordinary bovine milk. Especially the presence of sialylated and fucosylated oligosaccharides contributes to its health and development promoting features for newborn infants. [1] Nevertheless, not all newborns and especially premature infants sometimes cannot be breast fed for different reasons. For those children it is important that they receive a proper balanced formula product containing the above mentioned human milk oligosaccharides (HMOs). With respect to this we are developing new enzymatic routes for synthesis of sialylated and fucosylated oligosaccharides, which can be used as functional ingredient for infant formula. In a previous work two candidate hexoasaminidases (both belonging to the GH20 family) were identified from a metagenomic library, which were able to synthesize the basic HMO backbone structure, Lacto-N-triose II, from chitobiose and lactose by trans-glycosylation. [2] Since the yields using these enzymes were low (2% for hex1 and 8% for hex2 based on the donor substrate chitobiose) we wanted to increase their trans-glycosylation activity to increase their applicability for a feasible process. It was decided to follow a rational design approach first to keep the screening effort low. Therefore peptide pattern recognition (PPR) [3] analysis was performed on the whole GH20 CAZy family (approx. 3000 sequences) to identify other enzymes with potential trans-glycosylation activity based on relatedness. By phylogenetic analysis of the group containing the two known enzymes (approx. 1000 sequences) and subsequent alignment of the closely related sequences a loop insertion close to the active site was identified. Homology modelling revealed that introduction of this loop structure into hex1 and hex2 would lead to a significantly narrower active site and therefore contribute to exclusion of water from the active site, which is a well-known strategy to increase trans-glycosylation activity. The proposed loop mutants were then expressed, purified and characterized towards trans-glycosylation activity. For hex2 it turned out that none of the loop mutants showed an improved trans-glycosylation activity compared to the wild-type. But for hex1 three out of four showed an up to seven-fold improved trans-glycosylation activity compared to the wild-type, which refers even to a higher trans-glycosylation activity than previously observed for the hex2 wild-type. [4] In conclusion we succeeded in engineering an enzyme towards increased trans-glycosylation activity using a custom-made rational approach utilizing available sequence analysis methods. [1] L. Bode, Glycobiology 2012, 22, 1147–1162. [2] C. Nyffenegger, R. T. Nordvang, B. Zeuner, M. Łężyk, E. Difilippo, M. J. Logtenberg, H. A. Schols, A. S. Meyer, J. D. Mikkelsen, Appl. Microbiol. Biotechnol. 2015, 99, 7997–8009. [3] P. K. Busk, L. Lange, Appl. Environ. Microbiol. 2013, 79, 3380–3391. [4] S. B. Jamek, J. Muschiol, J. Holck, P. K. Busk, L. Lange, J. D. Mikkelsen, A. S. Meyer, 2017, manuscript submitted

Identification of Genomic Regions Associated with Phenotypic Variation between Dog Breeds using Selection Mapping

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Loop protein engineering for improved transglycosylation activity of a β‐N Acetylhexosaminidase

Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β‐N‐acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto‐N‐triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild‐type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1GTEPG, had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild‐type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate‐binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1GTEPG

Apoptotic cell death in disease-Current understanding of the NCCD 2023

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease

Targeting Tumour Hypoxia to Prevent Cancer Metastasis. From Biology, Biosensing and Technology to Drug Development: the METOXIA Consortium

Abstract The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia