Elucidation of different cold-adapted Atlantic cod (Gadus morhua) trypsin X isoenzymes

Trypsins from Atlantic cod (Gadus morhua), consisting of several isoenzymes, are highly active cold-adapted serine proteases. These trypsins are isolated for biomedical use in an eco-friendly manner from underutilized seafood by-products. Our group has explored the biochemical properties of trypsins and their high potential in biomedicine. For broader utilization of cod trypsins, further characterization of biochemical properties of the individual cod trypsin isoenzymes is of importance. For that purpose, a benzamidine purified trypsin isolate from Atlantic cod was analyzed. Anion exchange chromatography revealed eight peaks containing proteins around 24 kDa with tryptic activity. Based on mass spectrometric analysis, one isoenzyme gave the best match to cod trypsin I and six isoenzymes gave the best match to cod trypsin X. Amino terminal sequencing of two of these six trypsin isoenzymes showed identity to cod trypsin X. Three sequence variants of trypsin X were identified by cDNA analysis demonstrating that various forms of this enzyme exist. One trypsin X isoenzyme was selected for further characterization based on abundance and stability. Stepwise increase in catalytic efficiency (kcat/Km) of this trypsin X isoenzyme was obtained with substrates containing one to three amino acid residues. The study demonstrates that the catalytic efficiency of this trypsin X isoenzyme is comparable to that of cod trypsin I, the most abundant and highly active isoenzyme in the benzamidine cod trypsin isolate. Differences in pH stability and sensitivity to inhibitors of the trypsin X isoenzyme compared to cod trypsin I were detected that may be important for practical use.Research funded by AVS R&D Fund of Ministry of Fisheries and Agriculture in Iceland (R069-08, R11 028-11, R14 044-14) | Science and Technology Development Fund (120852-0611, 131804-0611)Ritrýnt tímaritPeer ReviewedPre-Prin

Nitroxide-labeled pyrimidines for non-covalent spin-labeling of abasic sites in DNA and RNA duplexes

Post-print (lokagerð höfundar)Non-covalent and site-directed spin labeling gives easy access to spin-labeled nucleic acids for the study of their structure and dynamics by electron paramagnetic resonance (EPR) spectroscopy. In a search for improved spin labels for non-covalent binding to abasic sites in duplex DNA and RNA, ten pyrimidine-derived spin labels were prepared in good yields and their binding was evaluated by continuous wave (CW)-EPR spectroscopy. Most of the spin labels showed lower binding affinity than the previously reported label c towards abasic sites in DNA and RNA. The most promising labels were triazole-linked spin labels and a pyrrolocytosine label. In particular, the N1-ethylamino derivative of a triazole-linked uracil spin label binds fully to both DNA and RNA containing an abasic site. This is the first example of a spin label that binds fully through non-covalent interactions with an abasic site in RNA.We thank the Icelandic Research Fund (110035022) and the University of Iceland Research Fund for financial support. S. A. S. acknowledges a doctoral fellowship from the University of Iceland Research Fund. We thank Dr S. Jonsdottir for assistance in collecting analytical data for structural characterization of the compounds that were prepared and members of the Sigurdsson research group for critical reading of the manuscript.Peer reviewe

Biochemical characterization of a native group III trypsin ZT from Atlantic cod (Gadus morhua)

Publisher's version (útgefin grein)Atlantic cod trypsin ZT is biochemically characterized for the first time in this report in comparison to a group I trypsin (cod trypsin I). To our knowledge, trypsin ZT is the first thoroughly characterized group III trypsin. A more detailed understanding of trypsin ZT biochemistry may give insight into its physiological role as well as its potential use within the biotechnology sector. Stability is an important factor when it comes to practical applications of enzymes. Compared to trypsin I, trypsin ZT shows differences in pH and heat stability, sensitivity to inhibitors and sub-site substrate specificity as shown by multiplex substrate profiling analysis. Based on the analysis, trypsin ZT cleaved at arginine and lysine as other trypsins. Furthermore, trypsin ZT is better than trypsin I in cleaving peptides containing several consecutive positively charged residues. Lysine- and arginine-rich amino acid sequences are frequently found in human viral proteins. Thus, trypsin ZT may be effective in inactivating human and fish viruses implying a possible role for the enzyme in the natural defence of Atlantic cod. The results from this study can lead to multiple practical applications of trypsin ZT.This work was supported by the AVS R&D Fund of Ministry of Fisheries and Agriculture in Iceland [grant reference number: R15 046-15 , R069-08 , R11 028-11 and R14 044-14 ]; and Technology Development Fund (The Icelandic Centre for Research ) [grant reference number: 120852-0611 and 131804-0611 ]. The funding source had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication.Peer Reviewe

Trypsín úr Atlantshafsþorski (Gadus morhua) – margbreytileiki ísóensíma og veiruhemjandi virkni

The aim of the thesis work was to identify and characterise different trypsin isoenzymes from a benzamidine-purified Atlantic cod (Gadus morhua) trypsin isolate. Another aim was to test the ability of cod trypsin to deactivate upper respiratory tract viruses. Initially, the stability of the trypsin isoenzymes I, X and ZT, in terms of temperature and pH, was characterised along with their sensitivity to various inhibitors. Multiplex substrate profiling by mass spectrometry of trypsin ZT and trypsin I revealed a difference in substrate specificity between the two trypsin isoenzymes. Furthermore, trypsin X, I and ZT showed a difference in sensitivity to inhibitors. The deactivation potential of the cod trypsin isolate was tested on several upper respiratory tract viruses. The results demonstrated that cod trypsin can deactivate rhinovirus A and coxsackievirus B. Furthermore, cod trypsin deactivated coronavirus. This was shown by using a Renilla luciferase assay and confirmed with a viability test. Furthermore, Western blot analysis showed that cod trypsin degraded recombinant coronavirus spike protein. The SARS coronavirus spike protein was treated with cod trypsin isoenzymes I and ZT to test the contribution of each isoenzyme to the cleavage. Both isoenzymes cleaved the SARS coronavirus spike protein at low trypsin concentrations. In conclusion, the results show that cod trypsin X and cod trypsin ZT have interesting characteristics differing from the previously characterised cod trypsin I. Cod trypsin was also shown to deactivate different viruses and to cleave recombinant coronavirus spike proteins.Markmið doktorsverkefnisins var að einangra og skilgreina mismunandi trypsín ísóensím úr bensamidín hreinsaðri trypsínblöndu. Jafnframt að rannsaka hvort þorskatrypsín geti óvirkjað veirur sem valda sýkingum í efri öndunarvegi. Í grein I og II var byrjað á að greina trypsín isóensímin X og ZT úr benzamidine hreinsaðri trypsín blöndu. Stöðugleiki þeirra gagnvart breytingum á hitastigi, sýrustigi og ensímhindrum var rannsakaður og borinn saman við áður þekkt trypsín I. Notuð var nýstárleg aðferð, sértæk hvarfefnamæling með massagreiningu, til að bera saman sérhæfni trypsín ísóensíma I og ZT en niðurstöðurnar leiddu í ljós umtalsverðan mun. Þá var einnig nokkur breytileiki í bindingu við ensímhindra á milli trypsín ísóensíma I, X og ZT. Eiginleiki trypsínblöndunnar til að óvirkja veirur, sem sýkja frumur í efri öndunarvegi var rannsakaður. Niðurstöðurnar leiddu í ljós að trypsínblandan óvirkjar rhinoveiru A og coxsackieveiru B. Meðhöndlun með lágum styrk af þorskatrypsíni leiddi einnig til óvirkjunar coronaveiru. Sýnt var fram á þennan eiginleika þorskatrypsíns með notkun Renilla luciferase mælingu ásamt mælingum á lífvænleika veirumeðhöndlaðra fruma. Þá var sýnt fram á getu þorskatrypsínblöndunnar við að kljúfa coronaveiru spike prótein með Western blot aðferð. Hæfni trypsín ísóensíma I og ZT við að brjóta niður SARS spike prótein var sambærileg byggt á SDS-PAGE greiningu. Niðurstöðurnar sýna að þorskatrypsín ísóensím X og ZT hafa áhugaverða eiginleika sem eru frábrugðnir eiginleikum trypsíns I, sem áður hefur verið skilgreint. Einnig var sýnt fram á að þorskatrypsín getur óvirkjað mismunandi öndunarfæraveirur og brotið niður spike prótein sem finnast á yfirborði coronaveira, þar með talið SARS veira

Nitroxide-derived pyrimidines for noncovalent spin-labeling of nucleic acids

To understand the function of nucleic acids in biological systems, their structural information is important. Electron paramagnetic resonance (EPR) spectroscopy has been increasingly used to study spin-labeled nucleic acids. Spin labels are usually incorporated site-specifically through covalent bonding, which can be difficult and time consuming. A new method was previously developed in our lab using noncovalent interactions where a spin label binds to an abasic site in a duplex DNA at low temperatures. However, the spin label (ҫ) that was used has a lengthy synthetic route and limited solubility in aqueous solutions. To search for spin labels with higher affinity for abasic sites and easier synthesis we have incorporated several nitroxides into the 5-position of pyrimidines. Two were made by an azide-alkyne Huisgen-Meldal-Sharpless (3+2) cycloaddition reaction (click reaction) and another two using palladium-catalyzed Sonogashira coupling. Among the click spin-labels, a U-analogue was fully bound at -30 °C while ca. 60% of a C-analogue bound to a duplex DNA containing an abasic site when placed opposite to A and G, respectively. However, 5-alkyne-linked pyrimidine spin-labeled derivatives of U and C bind only ca. 30% and 90% when paired with A and G, respectively. A C-analogue prepared by coupling 4-amino-TEMPO to 4-TPS U-derivative did not bind at all. Combined, our results indicate that stacking interactions contribute significantly to noncovalent binding of spin-labels at abasic sites in duplex DNA. Further work involving conjugation of a polyamine linker and an intercalator to the 1-position of the pyrimidines was initiated