Specificity of a β-porphyranase produced by the carrageenophyte red alga Chondrus crispus and implications of this unexpected activity on red algal biology
2022; Elsevier BV; Volume: 298; Issue: 12 Linguagem: Inglês
10.1016/j.jbc.2022.102707
ISSN1083-351X
AutoresGuillaume Manat, Mathieu Fanuel, Diane Jouanneau, Murielle Jam, Jessica Mac-Béar, Hélène Rogniaux, Théo Mora, Robert Larocque, Agnieszka P. Lipinska, Mirjam Czjzek, David Ropartz, E. Ficko-Blean,
Tópico(s)Marine and coastal plant biology
ResumoThe carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial β-carrageenase and β-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected β-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0–9.0). CcGH16-3 has a KM of 4.0 ± 0.8 μM, a kcat of 79.9 ± 6.9 s−1, and a kcat/KM of 20.1 ± 1.7 μM−1 s−1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its −1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This β-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization. The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial β-carrageenase and β-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected β-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0–9.0). CcGH16-3 has a KM of 4.0 ± 0.8 μM, a kcat of 79.9 ± 6.9 s−1, and a kcat/KM of 20.1 ± 1.7 μM−1 s−1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its −1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This β-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization. The macroalgal extracellular matrix (ECM) supports many functions such as protection against osmotic stress and desiccation, macroalgal flexibility, heavy metals bioabsorption, and is a barrier against pathogens (1Kloareg B. Quatrano R.S. Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides.Oceanogr. Mar. Biol. Annu. Rev. 1988; 26: 259-315Google Scholar, 2Kloareg B. Badis Y. Cock J.M. Michel G. Role and evolution of the extracellular matrix in the acquisition of complex multicellularity in eukaryotes: a macroalgal perspective.Genes (Basel). 2021; 12: 1059Crossref PubMed Scopus (19) Google Scholar). The major components of macroalgal ECMs are polysaccharides, which can have a large diversity of structures, even in the same species. Although algal polysaccharides have had major advances in their structural characterization, little data are available about macroalgal ECM interactions including biosynthesis, modification, and recycling. Most studies characterizing macroalgal ECM interactions have used WT enzymes. Two enzymatic activities were characterized in multicellular green algae, namely xyloglucan:xyloglucan endotransglucosylase (XET) and mixed-linkage-glucan:xyloglucan endotransglucosylase (MXE), activities that function in cell wall extension and remodeling (3Van Sandt V.S. Stieperaere H. Guisez Y. Verbelen J.P. Vissenberg K. XET activity is found near sites of growth and cell elongation in bryophytes and some green algae: new insights into the evolution of primary cell wall elongation.Ann. Bot. 2007; 99: 39-51Crossref PubMed Scopus (48) Google Scholar, 4Fry S.C. Mohler K.E. Nesselrode B.H. Frankova L. Mixed-linkage β-glucan: Xyloglucan endotransglucosylase, a novel wall-remodelling enzyme from Equisetum (horsetails) and charophytic algae.Plant J. 2008; 55: 240-252Crossref PubMed Scopus (98) Google Scholar, 5Herburger K. Ryan L.M. Popper Z.A. Holzinger A. Localisation and substrate specificities of transglycanases in charophyte algae relate to development and morphology.J. Cell Sci. 2018; 131jcs203208PubMed Google Scholar). A mannuronan C5-epimerase activity was demonstrated from protoplasts of the brown macroalga Laminaria digitata (6Nyvall P. Corre E. Boisset C. Barbeyron T. Rousvoal S. Scornet D. et al.Characterization of mannuronan C-5-epimerase genes from the brown alga Laminaria digitata.Plant Physiol. 2003; 133: 726-735Crossref PubMed Scopus (72) Google Scholar). In red macroalgae, galactose-sulfurylase enzymes catalyze the elimination of sulfate from galactose-6-sulfate or galactose-2,6-sulfate to form the 3,6-anhydro bridge (7Rhein-Knudsen N. Meyer A.S. Chemistry, gelation, and enzymatic modification of seaweed food hydrocolloids.Trends Food Sci. Tech. 2021; 109: 608-621Crossref Scopus (19) Google Scholar). Galactose-sulfurylase activity was first demonstrated on porphyran (a precursor for agar) using enzyme extracted from Porphyra umbilicalis (8Rees D.A. Enzymic synthesis of 3,6-anhydro-L-galactose within porphyran from L-galactose 6-sulphate units.Biochem. J. 1961; 81: 347-352Crossref PubMed Google Scholar). Two galactose-sulfurylase enzymes were purified from Chondrus crispus, galactose-sulfurylase I and galactose-sulfurylase II, and subsequently characterized as forming the 3,6-anhydro-d-galactose moiety in carrageenan (9Genicot-Joncour S. Poinas A. Richard O. Potin P. Rudolph B. Kloareg B. et al.The cyclization of the 3,6-anhydro-galactose ring of ι-carrageenan is catalyzed by two D-galactose-2,6-sulfurylases in the red alga Chondrus crispus.Plant Physiol. 2009; 151: 1609-1616Crossref PubMed Scopus (47) Google Scholar). Even fewer biochemical studies have been carried out on recombinant macroalgal ECM-active enzymes; this is presumably because protein production is challenging and the acidically charged glycan substrates are highly complex. Nevertheless, a recombinant brown algal mannuronan C5-epimerase, involved in alginate biosynthesis, was successfully refolded to produce active enzyme (10Fischl R. Bertelsen K. Gaillard F. Coelho S. Michel G. Klinger M. et al.The cell-wall active mannuronan C5-epimerases in the model brown alga Ectocarpus: from gene context to recombinant protein.Glycobiology. 2016; 26: 973-983Crossref PubMed Scopus (27) Google Scholar). In addition, two alginate lyases have been biochemically characterized, one from a red macroalga (11Inoue A. Mashino C. Uji T. Saga N. Mikami K. Ojima T. Characterization of an eukaryotic PL-7 alginate lyase in the marine red alga Pyropia yezoensis.Curr. Biotechnol. 2015; 4: 240-248Crossref PubMed Google Scholar), where alginate is not a known component, and the other from a brown macroalga (12Inoue A. Ojima T. Functional identification of alginate lyase from the brown alga Saccharina japonica.Sci. Rep. 2019; 9: 4937Crossref PubMed Scopus (31) Google Scholar). C. crispus is a carrageenophyte red macroalgae (Rhodophyta, Gigartinales) found along the northern Atlantic coast (Europe and America) that is mainly used in food and cosmetic industry for the properties of its algal ECM (13McHugh D. A Guide to the Seaweed Industry: FAO Fisheries Technical Paper. Food and Agriculture Organization of the United Nations, Rome2003: 61-72Google Scholar). The C. crispus ECM contains carrageenans that are linear, sulfated galactans with hybrid compositions; the basic unit is a D-galactose (G) disaccharide with alternating β-1,4 and α-1,3 linkages that is modified by sulfations on the disaccharide unit (14Ficko-Blean E. Hervé C. Michel G. Sweet and sour sugars from the sea: the biosynthesis and remodeling of sulfated cell wall polysaccharides from marine macroalgae.PiP. 2015; 2: 51-64Crossref Google Scholar). Another important modification is the unique bicyclic sugar α-3,6-anhydro-D-galactose (DA) (14Ficko-Blean E. Hervé C. Michel G. Sweet and sour sugars from the sea: the biosynthesis and remodeling of sulfated cell wall polysaccharides from marine macroalgae.PiP. 2015; 2: 51-64Crossref Google Scholar). Its biosynthesis was characterized using WT galactose-sulfurylases, the enzymatic desulfation of the C6 hydroxyl leads to the formation of the 3,6-anhydro-bridge (9Genicot-Joncour S. Poinas A. Richard O. Potin P. Rudolph B. Kloareg B. et al.The cyclization of the 3,6-anhydro-galactose ring of ι-carrageenan is catalyzed by two D-galactose-2,6-sulfurylases in the red alga Chondrus crispus.Plant Physiol. 2009; 151: 1609-1616Crossref PubMed Scopus (47) Google Scholar, 15Wong K.F. Craigie J.S. Sulfohydrolase activity and carrageenan biosynthesis in Chondrus crispus (Rhodophyceae).Plant Physiol. 1978; 61: 663-666Crossref PubMed Google Scholar). C. crispus has a complex isomorphic haplodiplontic life cycle. In gametophytes (n), the major carrageenan structures are the κ and ι-carrageenans and the minor biosynthetic precursors are μ- and ν-carrageenans (16Tasende M.G. Cid M. Fraga I.M. Spatial and temporal variations of Chondrus crispus (Gigartinaceae, Rhodophyta) carrageenan content in natural populations from Galicia (NW Spain).J. Appl. Phycol. 2012; 24: 941-951Crossref Scopus (17) Google Scholar, 17Chopin T. Floch J.Y. Ecophysiological and biochemical-study of 2 of the most contrasting forms of Chondrus crispus (Rhodophyta, Gigartinales).Mar. Ecol. Prog. Ser. 1992; 81: 185-195Crossref Scopus (24) Google Scholar). In tetrasporophytes (2n), there is only λ-carrageenan that has been described in the literature (18McCandless E.L. Craigie J.S. Walter J.A. Carrageenans in gametophytic and sporophytic stages of Chondrus crispus.Planta. 1973; 112: 201-212Crossref PubMed Scopus (94) Google Scholar). The gametophyte and tetrasporophyte life stages of C. crispus are isomorphic in the absence of reproductive structures; this suggests that the carrageenan structure may be important for the physiological differentiation between life stages of C. crispus (19Lipinska A.P. Collen J. Krueger-Hadfield S.A. Mora T. Ficko-Blean E. To gel or not to gel: differential expression of carrageenan-related genes between the gametophyte and tetasporophyte life cycle stages of the red alga Chondrus crispus.Sci. Rep. 2020; 10: 11498Crossref PubMed Scopus (14) Google Scholar). This is supported by the susceptibility of tetrasporophytes, but not gametophytes, of C. crispus to a green algal pathogen, Ulvella (Acrochaete) operculata (20Correa J.A. Mclachlan J.L. Endophytic algae of Chondrus crispus (Rhodophyta). III. Host specificity.J. Phycol. 1991; 27: 448-459Crossref Scopus (78) Google Scholar, 21Krueger-Hadfield S.A. Population structure in the haploid-diploid red alga Chondrus crispus: Mating system, genetic differentiation and epidemiology. UPMC Paris 6 with l'Universidad católica de Chile, 2011Google Scholar). When artificially introduced to the milieu, λ-carrageenan oligosaccharides induced increased virulence toward gametophytes and κ-carrageenan oligosaccharides reduced virulence toward tetrasporopytes by the green algal pathogen (22Bouarab K. Potin P. Correa J. Kloareg B. Sulfated oligosaccharides mediate the interaction between a marine red alga and its green algal pathogenic endophyte.Plant Cell. 1999; 11: 1635-1650Crossref PubMed Scopus (113) Google Scholar). Thus, life cycle specific carrageenan metabolites have functionally distinct biological signaling properties and influence susceptibility to U. operculata. The genome sequencing and annotation of C. crispus has led to the identification of three genes (ccgh16-1, ccgh16-2, and ccgh16-3) coding for CAZy (Carbohydrate Active Enzymes database, URL http://www.cazy.org/) GH16 family members (CcGH16-1, CcGH16-2, and CcGH16-3, Fig. 1 and Table S1) (23Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42: D490-D495Crossref PubMed Scopus (4237) Google Scholar, 24Collen J. Porcel B. Carre W. Ball S.G. Chaparro C. Tonon T. et al.Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 5247-5252Crossref PubMed Scopus (252) Google Scholar). The differences in carrageenan composition between the isomorphic life stages of C. crispus suggest that genes involved in carrageenan biosynthesis may be differentially expressed between these life stages. The gene ccgh16-1 was silent in the tetrasporophyte and gametophyte stages under the conditions assayed. On the other hand, the genes ccgh16-2 and ccgh16-3 were expressed in all the life cycle stages, with a significant increase of ccgh16-3 expression in male gametophytes and tetrasporophytes relative to the female gametophytes (19Lipinska A.P. Collen J. Krueger-Hadfield S.A. Mora T. Ficko-Blean E. To gel or not to gel: differential expression of carrageenan-related genes between the gametophyte and tetasporophyte life cycle stages of the red alga Chondrus crispus.Sci. Rep. 2020; 10: 11498Crossref PubMed Scopus (14) Google Scholar). Two variants of 'typical' active sites within the GH16 family exist with the ExDxE and ExDxxE conserved amino acid motifs that are clearly phylogenetically separated (25Viborg A.H. Terrapon N. Lombard V. Michel G. Czjzek M. Henrissat B. et al.A subfamily roadmap of the evolutionarily diverse glycoside hydrolase family 16 (GH16).J. Biol. Chem. 2019; 294: 15973-15986Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 26Brawley S.H. Blouin N.A. Ficko-Blean E. Wheeler G.L. Lohr M. Goodson H.V. et al.Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E6361-E6370Crossref PubMed Scopus (158) Google Scholar, 27Barbeyron T. Gerard A. Potin P. Henrissat B. Kloareg B. The κ-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases.Mol. Biol. Evol. 1998; 15: 528-537Crossref PubMed Scopus (99) Google Scholar). In the first motif, the hyaluronidase (28Bakke M. Kamei J. Obata A. Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp.FEBS Lett. 2011; 585: 115-120Crossref PubMed Scopus (13) Google Scholar), laminarinase (29Labourel A. Jam M. Jeudy A. Hehemann J.H. Czjzek M. Michel G. The β-glucanase ZgLamA from Zobellia galactanivorans evolved a bent active site adapted for efficient degradation of algal laminarin.J. Biol. Chem. 2014; 289: 2027-2042Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), β-1,3-galactanase (30Kotake T. Hirata N. Degi Y. Ishiguro M. Kitazawa K. Takata R. et al.Endo-β-1,3-galactanase from winter mushroom Flammulina velutipes.J. Biol. Chem. 2011; 286: 27848-27854Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), β-1,3/β-1,4-glucanase (31Linton S.M. Cameron M.S. Gray M.C. Donald J.A. Saborowski R. von Bergen M. et al.A glycosyl hydrolase family 16 gene is responsible for the endogenous production of β-1,3-glucanases within decapod crustaceans.Gene. 2015; 569: 203-217Crossref PubMed Scopus (13) Google Scholar, 32Planas A. Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering.Biochim. Biophys. Acta. 2000; 1543: 361-382Crossref PubMed Scopus (236) Google Scholar), and xyloglucan endotransglucosylase/hydrolase (33Baumann M.J. Eklof J.M. Michel G. Kallas A.M. Teeri T.T. Czjzek M. et al.Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism.Plant Cell. 2007; 19: 1947-1963Crossref PubMed Scopus (206) Google Scholar) activities are defined. The second motif is possessed by enzymes from several bacteria that degrade the ECMs of red algae including β-agarases (34Jam M. Flament D. Allouch J. Potin P. Thion L. Kloareg B. et al.The endo-β-agarases AgaA and AgaB from the marine bacterium Zobellia galactanivorans: two paralogue enzymes with different molecular organizations and catalytic behaviours.Biochem. J. 2005; 385: 703-713Crossref PubMed Scopus (119) Google Scholar, 35Naretto A. Fanuel M. Ropartz D. Rogniaux H. Larocque R. Czjzek M. et al.The agar-specific hydrolase ZgAgaC from the marine bacterium Zobellia galactanivorans defines a new GH16 protein subfamily.J. Biol. Chem. 2019; 294: 6923-6939Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), β-porphyranases (36Hehemann J.H. Correc G. Thomas F. Bernard T. Barbeyron T. Jam M. et al.Biochemical and structural characterization of the complex agarolytic enzyme system from the marine bacterium Zobellia galactanivorans.J. Biol. Chem. 2012; 287: 30571-30584Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and κ- or β-carrageenanases (all β-1,4-galactanases) (37Barbeyron T. Henrissat B. Kloareg B. The gene encoding the κ-carrageenase of Alteromonas carrageenovora is related to β-1,3-1,4-glucanases.Gene. 1994; 139: 105-109Crossref PubMed Scopus (56) Google Scholar, 38Schultz-Johansen M. Bech P.K. Hennessy R.C. Glaring M.A. Barbeyron T. Czjzek M. et al.A novel enzyme portfolio for red algal polysaccharide degradation in the marine bacterium Paraglaciecola hydrolytica S66(T) encoded in a sizeable polysaccharide utilization locus.Front. Microbiol. 2018; 9: 839Crossref PubMed Scopus (46) Google Scholar, 39Matard-Mann M. Bernard T. Leroux C. Barbeyron T. Larocque R. Prechoux A. et al.Structural insights into marine carbohydrate degradation by family GH16-carrageenases.J. Biol. Chem. 2017; 292: 19919-19934Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). All of the C. crispus GH16 enzymes belong to the second, ExDxxE clade shared by GH16 members from both carrageenophyte and agarophyte red algae (25Viborg A.H. Terrapon N. Lombard V. Michel G. Czjzek M. Henrissat B. et al.A subfamily roadmap of the evolutionarily diverse glycoside hydrolase family 16 (GH16).J. Biol. Chem. 2019; 294: 15973-15986Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 26Brawley S.H. Blouin N.A. Ficko-Blean E. Wheeler G.L. Lohr M. Goodson H.V. et al.Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E6361-E6370Crossref PubMed Scopus (158) Google Scholar, 27Barbeyron T. Gerard A. Potin P. Henrissat B. Kloareg B. The κ-carrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases.Mol. Biol. Evol. 1998; 15: 528-537Crossref PubMed Scopus (99) Google Scholar). The similarity was supported by a protein sequence and structure similarity network analysis (25Viborg A.H. Terrapon N. Lombard V. Michel G. Czjzek M. Henrissat B. et al.A subfamily roadmap of the evolutionarily diverse glycoside hydrolase family 16 (GH16).J. Biol. Chem. 2019; 294: 15973-15986Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and phylogenetic analyses of the GH16 family (26Brawley S.H. Blouin N.A. Ficko-Blean E. Wheeler G.L. Lohr M. Goodson H.V. et al.Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E6361-E6370Crossref PubMed Scopus (158) Google Scholar). These studies revealed that the C. crispus GH16 enzymes form a subclade together with the red algal GH16 enzymes from P. umbilicalis. Moreover, close neighboring clades were shown to contain biochemically characterized bacterial β-porphyranases and β-agarases (GH16 subfamilies 11, 12, 13, 14, 16, and 26) (25Viborg A.H. Terrapon N. Lombard V. Michel G. Czjzek M. Henrissat B. et al.A subfamily roadmap of the evolutionarily diverse glycoside hydrolase family 16 (GH16).J. Biol. Chem. 2019; 294: 15973-15986Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 26Brawley S.H. Blouin N.A. Ficko-Blean E. Wheeler G.L. Lohr M. Goodson H.V. et al.Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E6361-E6370Crossref PubMed Scopus (158) Google Scholar). It was initially hypothesized that the three CcGH16 proteins identified may be involved in carrageenan biosynthesis, remodeling, and/or recycling, based on the carrageenan composition of the ECM of C. crispus and the CcGH16 enzyme's phylogenetic relationship with marine bacterial sulfated polysaccharide degrading enzymes (19Lipinska A.P. Collen J. Krueger-Hadfield S.A. Mora T. Ficko-Blean E. To gel or not to gel: differential expression of carrageenan-related genes between the gametophyte and tetasporophyte life cycle stages of the red alga Chondrus crispus.Sci. Rep. 2020; 10: 11498Crossref PubMed Scopus (14) Google Scholar). Here, we investigate the biochemistry of the red algal CcGH16-3 enzyme, which was found to have an unexpected β-porphyranase activity. This investigation provides one of only a couple biochemical studies on recombinant red algal glycoside hydrolases and brings a new perspective to glycoside hydrolase function in seaweed. In the carrageenophyte red alga C. crispus, three different CcGH16 enzymes (1 - CDF40276.1, 2 - CDF41280.1, and 3 - CDF33251.1) were identified with 469 (52.6 kDa), 301 (35.8 kDa), and 262 (29.9 kDa) amino acids, respectively, and sharing between 30% and 34% sequence identity (Fig. 1). One GH16 gene in particular, ccgh16-3, was previously identified (19Lipinska A.P. Collen J. Krueger-Hadfield S.A. Mora T. Ficko-Blean E. To gel or not to gel: differential expression of carrageenan-related genes between the gametophyte and tetasporophyte life cycle stages of the red alga Chondrus crispus.Sci. Rep. 2020; 10: 11498Crossref PubMed Scopus (14) Google Scholar) as differentially regulated between the stages of the complex isomorphic haplodiplontic life cycle of C. crispus (40Collen J. Cornish M.L. Craigie J. Ficko-Blean E. Herve C. Krueger-Hadfield S.A. et al.Chondrus crispus - A Present and Historical Model Organism for Red Seaweeds. Elsevier, Paris2014Crossref Scopus (31) Google Scholar). CcGH16-3 amino acid BLAST revealed the closest GH16 homologs in other red algae were from agarophyte red algae (Porphyra, Gracilariopsis) with 33% to 48% sequence identity but this result could be explained by the weakness of the red algal genome database and the distinct lack of carrageenophyte red algal genomes (with the sole exception at the time of writing of C. crispus). Phylogenetic analyses revealed that the CcGH16 enzymes cluster closest first with uncharacterized marine red algal and then bacterial GH16 enzymes including β-porphyranases and β-agarases (26Brawley S.H. Blouin N.A. Ficko-Blean E. Wheeler G.L. Lohr M. Goodson H.V. et al.Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E6361-E6370Crossref PubMed Scopus (158) Google Scholar). Since porphyran is not a known component of the C. crispus ECM, this led to the hypothesis that the C. crispus GH16 enzymes are active as carrageenases and involved in ECM modification. Furthermore, the ccgh16-3 gene from C. crispus demonstrates differential gene expression between tetrasporophytes relative to female gametophytes; these are multicellular life stages that have different carrageenan content (19Lipinska A.P. Collen J. Krueger-Hadfield S.A. Mora T. Ficko-Blean E. To gel or not to gel: differential expression of carrageenan-related genes between the gametophyte and tetasporophyte life cycle stages of the red alga Chondrus crispus.Sci. Rep. 2020; 10: 11498Crossref PubMed Scopus (14) Google Scholar). Overall, this suggested that the C. crispus CcGH16 enzymes may be involved in red algal sulfated ECM polysaccharide modification. The annotation of the genome of C. crispus initially identified the gh16 genes in the alga (24Collen J. Porcel B. Carre W. Ball S.G. Chaparro C. Tonon T. et al.Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 5247-5252Crossref PubMed Scopus (252) Google Scholar). RNA-seq analysis further detected differential expression of the ccgh16-3 gene (19Lipinska A.P. Collen J. Krueger-Hadfield S.A. Mora T. Ficko-Blean E. To gel or not to gel: differential expression of carrageenan-related genes between the gametophyte and tetasporophyte life cycle stages of the red alga Chondrus crispus.Sci. Rep. 2020; 10: 11498Crossref PubMed Scopus (14) Google Scholar). However, due to the close phylogenetic relationship to bacterial GH16 enzymes, we thought it to be prudent to confirm that the ccgh16-3 gene is indeed located in the C. crispus genome and was not from bacterial contamination. In order to do so, we analyzed the context of the ccgh16-3 genetic locus (Fig. 2) and undertook a fine GH16 subfamily phylogenetic analysis (Fig. 3).Figure 3Phylogenetic analysis of the red algal GH16 enzymes (GH16_xx) shown in different shades of red depending on the red algal species (Table S1). Only GH16 catalytic modules were included in the analysis, not full-length sequences (Supporting Information File 1). Nearest subfamilies included in the analysis were chosen based on their amino acid sequence similarity to the red algal GH16 enzymes. Bootstraps above 40 are shown. Asterisks (∗) show characterized GH16 enzymes in the CAZy database (23Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42: D490-D495Crossref PubMed Scopus (4237) Google Scholar) and 3D indicates the structures have been determined. The more distantly related GH16 subfamily 3 laminarinases (GH16_3) were chosen as an outgroup.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Upstream of the ccgh16-3 gene of C. crispus, the gene chc_t00002034001 codes for a protein predicted by BLAST (41Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) to be the large subunit of the eukaryotic protein guanidine triphosphate phosphatase 1 (GTPase1). The closest identified non–red algal homologs of the GTPase1 protein are fungal: Tuber magnatum and Aspergillus chevalieri, which share 39.7% and 38.9% sequence identity over 79.0% query coverage. The closest characterized GTPase homologs are found in Arabidopsis thaliana, and in fungi, these GTPases are important for eukaryotic ribosomal biogenesis (42Weis B.L. Missbach S. Marzi J. Bohnsack M.T. Schleiff E. The 60S associated ribosome biogenesis factor LSG1-2 is required for 40S maturation in Arabidopsis thaliana.Plant J. 2014; 80: 1043-1056Crossref PubMed Scopus (37) Google Scholar, 43Kallstrom G. Hedges J. Johnson A. The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively.Mol. Cell. Biol. 2003; 23: 4344-4355Crossref PubMed Scopus (117) Google Scholar). The chc_t00002034001 gene is followed by chc_t00002035001, which has candidates in both bacteria and eukaryotes and is predicted to code for the redox protein peroxiredoxin. The chc_t00002035001 gene is followed by the ccgh16-3 gene (chc_t00009578001) (Fig. 2) (24Collen J. Porcel B. Carre W. Ball S.G. Chaparro C. Tonon T. et al.Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 5247-5252Crossref PubMed Scopus (252) Google Scholar). Using PCR, we were successfully able to amplify a 1.6 kb DNA fragment from C. crispus tetrasporophyte genomic DNA template located between the eukaryotic gene chc_t00002034001 and ccgh16-3 (Fig. 2). Sequencing confirmed the predicted sequences for the two target genes chc_t00002034001 and ccgh16-3. This result confirms that the ccgh16-3 gene is indeed found in the C. crispus genome and it further supports the genome assembly of C. crispus (24Collen J. Porcel B. Carre W. Ball S.G. Chaparro C. Tonon T. et al.Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 5247-5252Crossref PubMed Scopu
Referência(s)