The two oceans project : searching for novel carbohydrate sulfoconjugation enzymes

Prins, Ricardo (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Marine algae contain a wide range of carbohydrate-based bioactive compounds, which promote extra-nutritional health benefits. In marine algae, the most sought after bioactive compounds are sulfoconjugated polysaccharides (SPs) which have the potential to be used as functional ingredients in the nutraceuticals industry. SPs are of particular biological interest to human health and have been shown to exert anti-inflammatory, anti-tumorigenic, immunostimulatory, and more recently anti-viral activities. The specific biological properties of SPs are attributed to their monomer composition, sulfate content, and sulfoconjugation pattern present on the sugar backbone. The biochemical mechanisms through which these polysaccharides are sulfoconjugated are well characterised in animals and are catalysed by a superfamily of enzymes termed sulfotransferases (SULTs, EC 2.8.2). In vivo, SULTs are responsible for the enzymatic process termed sulfoconjugation. This entails the transfer of a sulfuryl group (SO3) from a donor molecule, classically 3’-phosphoadenosine 5’-phosphosulfate (PAPS), to an acceptor molecule e.g. alcohol, amine, sugar or phenolic compound. The current state-of-the-art involves the isolation and purification of sulfoconjugated compounds from marine organisms. These compounds are then tested for various bioactivities (Hamed et al., 2015; Wijesekara et al., 2011). In contrast, here we used a high-throughput RNA-seq approach to identify 18 putative SULT transcripts and subsequently isolated one carbohydrate SULT. Eight of the SULTs identified from the P. corallorhiza transcriptome had a Sulfotransfer_1 domain. However, four of the eight Sulfotransfer_1 SULTs also had a Sulfotransfer_3 domain. Additionally, seven SULTs had a Sulfotransfer_2 domain, with one also possessing a Gal-3-O_sulfotr domain. The SULTs identified with overlapping domains were not exclusively assigned to any one SULT family, as they had statistically significant homology (<0.05) to either the Sulfotransfer_1 and Sulfotransfer_3 or the Sulfotransfer_2 and the Gal-3-O_sulfotr domain containing SULT entries, respectively. Additionally, this approach included the diurnal sampling of a ubiquitously distributed red algal species, Plocamium corallorhiza, as a generic representative of the Rhodophyta phylum. A diurnal differential gene expression analysis was used to assess potential differences in gene expression, specifically with regard to galactan biosynthesis. Ultimately, 639 nocturnally differentially expressed genes (DEGs) were identified. However, no DEGs relating to galactan biosynthesis as it relates to starch and sucrose, galactose or sulfur metabolism pathways, were present within the 639 DEGs.

AFRIKAANSE OPSOMMING: Mariene alge bevat ‘n wye verskeidenheid van koolhidraat gebaseerde bioaktiewe verbindings wat meer as net voedingswaarde verskaf, maar ook gesondsheids- voordele inhou. Die mees gesogte bioaktiewe verbingings in mariene alge is, gesulfo-konjugeerde polisakkariede (SPs), wat potensieel as ‘n funksionele bestandeel in die gesondsheidsindustrie gebruik kan word. SPs besit anti-inflammatoriese, anti-gewas, immuun stimileerende eienskappe, en meer. Onlangs is anti-virale bioaktivitiet ook hiervoor bewys. Die bogenoemde bioaktiwiteite maak SPs van besonderse belangvir menslike gesondheid. Die spesifieke biologiese eienskappe van SPs word toegeskryf aan hul monomeer samestelling, sulfaat-inhoud en die gesulfo-konjugeerde patroon teenwoordig op die suiker-ruggraat. Die biochemiese meganismes waardeur hierdie polisakkariede gesulfo-konjugeered word, is goed nagevors in diere en word gekataliseer deur ‘n superfamilie van ensieme, naamlik sulfotransferases (SULT, EC 2.8.2). Die huidiglike benadering, behels die isolasie en suiwering van gesulfo-konjugeerde verbindings vanaf mariene organismes. Hierdie verbindings word dan getoets vir verskeie bio-aktiwiteite (Hamed et al., 2015; Wijesekara et al., 2011). Ons benadering poog om die ensieme, naamlik SULTs, wat verantwoordelik is vir sulfo-konjugering te identifiseer en te isoleer vanaf ‘n makro rooi alg. Hier was RNA-seq gebruik op Plocamium corallorhiza, ‘n algemene Suid-Afrikaanse rooi alg. Gevolglik was 18 SULTs geïdentifiseer en een koolhidraat SULT was geïsoleer. Agt van die SULTs geïdentifiseer uit die P. corallorhiza transkriptoom het 'n Sulfotransfer_1 domein. Alhoewel, vier van die agt Sulfotransfer_1 SULTs het ook 'n Sulfotransfer_3 domein. Daarbenewens, sewe SULTs het 'n Sulfotransfer_2 domein, met een wat ook in besit van 'n Gal-3-O_sulfotr domein. Die SULTs geïdentifiseer met oorvleuelende domeine behoort nie uitsluitlik net aan een familie nie, omdat die domeine het statistiese beduidende homologoë (<0,05) om óf die Sulfotransfer_1 en Sulfotransfer_3 of die Sulfotransfer_2 en die Gal-3-O_sulfotr domein inskrywings bevat, onderskeidelik. Daarbenewens, sluit hierdie benadering ‘n middag en middernag bemonstering in vir geenuitdrukking analises, spesifiek ten opsigte van galaktaan biosintese. Alhoewel, geen naglikse differensieele geenuitdrukking (DEGs) van belang tot galaktaan sintese, soos dit verband hou met stysel and sukrose, galaktose and swael metabolisme, geïdentifiseer was nie, is 639 DEGs wel geïdentifiseer.

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