Skip to main content
SpringerLink
Log in

Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1∆ null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abeijon C, Orlean P, Robbins PW, Hirschberg CB (1989) Topography of glycosylation in yeast: characterization of GDP-mannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles. Proc Natl Acad Sci USA 86:6935–6939

    Article  PubMed  CAS  Google Scholar 

  • Abeijon C, Yanagisawa K, Mandon EC, Häusler A, Moremen K, Hirschberg CB, Robbins PW (1993) Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. J Cell Biol 122:307–323

    Article  PubMed  CAS  Google Scholar 

  • Abeijon C, Robbins PW, Hirschberg CB (1996) Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis. Proc Natl Acad Sci USA 93:5963–5968

    Article  PubMed  CAS  Google Scholar 

  • Aquino-Piñero E, Rodríguez del Valle N (2002) Characterization of a protein kinase C gene in Sporothrix schenckii and its expression during the yeast-to-mycelium transition. Med Mycol 40:185–199

    PubMed  Google Scholar 

  • Bastos KP, Bailäo AM, Borges CL, Faria FP, Felipe MSS, Silva MG, Martins WS, Fiúza RB, Pereira M, Soares CMA (2007) The transcriptome analysis of early morphogenesis in Paracoccidioides brasiliensis mycelium reveals novel and induced genes potentially associated to the dimorphic process. BMC Microbiol 7:29–43

    Article  PubMed  Google Scholar 

  • Beaudoing E, Freier S, Wyatt JR, Claverie JM, Gautheret D (2000) Patterns of variant polyadenylation signal usage in human genes. Genome Res 10:1001–1010

    Article  PubMed  CAS  Google Scholar 

  • Berninsone P, Miret JJ, Hirschberg CB (1994) The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles. J Biol Chem 269:207–211

    PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Brandan E, Fleischer B (1982) Orientation and role of nucleoside diphosphatase and 5′-nucleotidase in Golgi vesicles from rat liver. Biochemistry 21:4640–4645

    Article  PubMed  CAS  Google Scholar 

  • Capasso JM, Hirschberg CB (1984) Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane. Proc Natl Acad Sci USA 81:7051–7055

    Article  PubMed  CAS  Google Scholar 

  • D’Alessio C, Trombetta ES, Parodi AJ (2003) Nucleoside diphosphatase and glycosyltransferase activities can localize to different subcellular compartments in Schizosaccharomyces pombe. J Biol Chem 278:22379–22387

    Article  PubMed  Google Scholar 

  • Davis CA, Grate L, Spingola M, Ares MJ (2000) Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast. Nucleic Acids Res 28:1700–1706

    Article  PubMed  CAS  Google Scholar 

  • De Angioletti M, Lacerra G, Sabato V, Carestia C (2004) β + 45 G → C: a novel silent β-thalassaemia mutation, the first in the Kozak sequence. British J Haematol 124:224–231

    Article  Google Scholar 

  • Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer ELL, Eddy SR, Bateman A (2009) The Pfam protein families database. Nucleic Acids Res 38:211–222

    Article  Google Scholar 

  • Hernández-Cervantes A, Mora-Montes HM, Alvarez-Vargas A, Díaz-Jiménez DF, Robledo-Ortiz CI, Flores-Carreón A (2012) Isolation of Sporothrix schenckii MNT1 and the biochemical and functional characterization of the encoded α1,2-mannosyltransferase activity. Microbiology 158(suppl1):D2419–D2427

    Article  Google Scholar 

  • Herrero AB, Uccelletti D, Hirschberg CB, Dominguez A, Abeijon C (2002) The Golgi GDPase of the fungal pathogen Candida albicans affects morphogenesis, glycosylation, and cell wall properties. Eukaryot Cell 1:420–431

    Article  PubMed  CAS  Google Scholar 

  • Hirschberg CB, Robbins PW, Abeijon C (1998) Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Ann Rev Biochem 67:49–69

    Article  PubMed  CAS  Google Scholar 

  • Jörgensen TR, Goosen T, van den Hondel CA, Ram AF, Iversen JJ (2009) Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway. BMC Genomics 10:44–59

    Article  PubMed  Google Scholar 

  • Juneau K, Miranda M, Hillenmeyer ME, Nislow C, Davis RW (2006) Introns regulate RNA and protein abundance in Yeast. Genetics 174:511–518

    Article  PubMed  CAS  Google Scholar 

  • Kozak M (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15:8125–8148

    Article  PubMed  CAS  Google Scholar 

  • Lopes-Alves LM, Mendonca-Previato L, Fournet B, Degand P, Previato JO (1992) O-glycosidically linked oligosaccharides from peptidorhamnomannans of Sporothrix schenckii. Glycoconj J 9:75–81

    Article  PubMed  CAS  Google Scholar 

  • Lopes-Bezerra LM, Schubach A, Costa RO (2006) Sporothrix schenckii and sporotrichosis. An Acad Bras Cienc 78:293–308

    Article  PubMed  Google Scholar 

  • Lopez-Avalos MD, Uccelletti D, Abeijon C, Hirschberg CB (2001) The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles. Glycobiology 11:413–422

    Article  PubMed  CAS  Google Scholar 

  • López-Romero E, Reyes-Montes MR, Pérez-Torres A, Ruiz-Baca E, Villagómez-Castro JC, Mora-Montes HM, Flores-Carreón A, Toriello C (2011) Sporothrix schenckii complex and sporotrichosis, an emerging health problem. Future Microbiology 6:85–102

    Article  PubMed  Google Scholar 

  • Lussier M, Sdicu AM, Bussey H (1999) The KTR and MNN1 mannosyltransferase families of Saccharomyces cerevisiae. Biochim Biophys Acta 1426:323–334

    Article  PubMed  CAS  Google Scholar 

  • Madrid IM, Xavier MO, Mattei AS, Fernandes CG, Guim TN, Santin R, Schuch LFD, Nobre MO, Araújo-Meireles MC (2010) Role of melanin in the pathogenesis of cutaneous sporotrichosis. Microbes Infect 12:162–165

    Article  PubMed  CAS  Google Scholar 

  • Milanesi L, Muselli M, Arrigo P (1996) Hamming-clustering method for signals prediction in 5′ and 3′ regions of eukaryotic genes. Comput Appl Biosci 12:399–404

    PubMed  CAS  Google Scholar 

  • Mora-Montes HM, Bates S, Netea MG, Díaz-Jiménez DF, López-Romero E, Samuel Z, Ponce-Noyola P, Kullberg BJ, Brown AJP, Odds FC, Flores-Carreón A, Gow NAR (2007) Endoplasmic reticulum alpha-glycosidases of Candida albicans are required for N-glycosylation, cell wall integrity, and normal host-fungus interaction. Eukaryot Cell 6:2184–2193

    Article  PubMed  CAS  Google Scholar 

  • Mora-Montes HM, Ponce-Noyola P, Villagómez-Castro JC, Gow NAR, Flores-Carreón A, López-Romero E (2009) Protein glycosylation in Candida. Future Microbiology 4:1167–1183

    Article  PubMed  CAS  Google Scholar 

  • Mora-Montes HM, Bates S, Netea MG, Castillo L, Brand A, Buurman ET, Díaz-Jiménez DF, Jan-Kullberg B, Brown AJ, Odds FC, Gow NAR (2010a) A Multifunctional Mannosyltransferase Family in Candida albicans determines cell wall mannan structure and host-fungus interactions. J Biol Chem 285:12087–12095

    Article  PubMed  CAS  Google Scholar 

  • Mora-Montes HM, Robledo-Ortiz CI, González-Sánchez LC, López-Esparza A, López-Romero E, Flores-Carreón A (2010b) Purification and biochemical characterisation of endoplasmic reticulum α1,2-mannosidase from Sporothrix schenckii. Mem Inst Oswaldo Cruz 105:79–85

    Article  PubMed  CAS  Google Scholar 

  • Pérez-Sánchez L, González E, Colón-Lorenzo EE, González-Velázquez W, González-Méndez R, Rodríguez-del Valle N (2010) Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay. BMC Microbiol 10:317–333

    Article  PubMed  Google Scholar 

  • Ramos-e-Silva M, Vasconcelos C, Carneiro S, Cestari T (2007) Sporotrichosis. Clin Dermatol 25:181–187

    Article  PubMed  Google Scholar 

  • Reed R, Maniatis T (1985) Intron sequences involved in lariat formation during pre-mRNA splicing. Cell 41:95–105

    Article  PubMed  CAS  Google Scholar 

  • Robledo-Ortiz CI, Flores-Carreón A, Hernández-Cervantes A, Alvarez-Vargas A, Lee KK, Díaz-Jiménez DF, Munro CA, Cano-Canchola C, Mora-Montes HM (2012) Isolation and functional characterisation of Sporothrix schenckii ROT2, the encoding gene for the endoplasmic reticulum glucosidase II. Fungal Biol 116:910–918

    Article  PubMed  CAS  Google Scholar 

  • Rogozin IB, Kochetov AV, Kondrashov FA, Koonin EV, Milanesi L (2001) Presence of ATG triplets in 5′ untranslated regions of eukaryotic cDNAs correlates with a ‘weak’ context of the start codon. Bioinformatics 17:890–900

    Article  PubMed  CAS  Google Scholar 

  • Ruiz-Baca E, Villagómez-Castro JC, Leal-Morales CA, Sabanero-López M, Flores-Carreón A, López-Romero E (2005) Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases. Antonie Van Leeuwenhoek 88:221–230

    Article  PubMed  CAS  Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics: a laboratory course manual. Cold spring harbor laboratory, Cold Spring Harbor

    Google Scholar 

  • Tehler A, Little DP, Farris JS (2003) The full-length phylogenetic tree from 1551 ribosomal sequences of chitinous fungi. Fungi Mycological Res 107:901–916

    Article  CAS  Google Scholar 

  • Torres-Guerrero H (1999) Ploidy study in Sporothrix schenkii. Fungal Gen Biol 27:49–54

    Article  CAS  Google Scholar 

  • Trachtulec Z, Forejt J (1999) Transcription and RNA processing of mammalian genes in Saccharomyces cerevisiae. Nucleic Acids Res 27:526–531

    Article  PubMed  CAS  Google Scholar 

  • Travassos LR, Lloyd KO (1980) Sporothrix schenckii and related species of Ceratocystis. Microbiol Rev 44:683–721

    PubMed  CAS  Google Scholar 

  • Travassos LR, de Sousa W, Mendonça-Previato L, Lloyd KO (1977) Location and biochemical nature of surface components reacting with concanavalin A in different cell types of Sporothrix schenckii. Exp Mycol 1:293–305

    Article  CAS  Google Scholar 

  • Valentín-Berrios S, González-Velázquez W, Pérez-Sánchez L, González-Méndez R, Rodríguez-del Valle N (2009) Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein alpha subunit in Sporothrix schenckii. BMC Microbiol 9:100–114

    Article  PubMed  Google Scholar 

  • Wang TF, Guidotti G (1998) Golgi localization and functional expression of human uridine diphosphatase. J Biol Chem 273:11392–11399

    Article  PubMed  CAS  Google Scholar 

  • Wulff C, Norambuena L, Orellana A (2000) GDP-fucose uptake into the Golgi apparatus during Xyloglucan biosynthesis requires the activity of a transporter-like protein other than the UDP-glucose transporter. Plant Physiol 122:867–878

    Article  PubMed  CAS  Google Scholar 

  • Yanagisawa K, Resnick D, Abeijon C, Robbins PW, Hirschberg CB (1990) A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization. J Biol Chem 265:19351–19355

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We wish to thank Professors Everardo López-Romero and Julio César Villagómez-Castro for their advice during the determination of enzyme activities and QFB Tannia Razo-Soria for their technical support. This work was supported by SEP-CONACyT CB-2007-01 Ref. No. 83414, México, and Universidad de Guanajuato.

Author information

Authors and Affiliations

  1. Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato, Noria Alta s/n, Col. Noria Alta, CP 36050, Guanajuato, Gto, Mexico

    Adolfo López-Esparza, Aurelio Álvarez-Vargas, Héctor M. Mora-Montes, Arturo Hernández-Cervantes, Ma. Del Carmen Cano-Canchola & Arturo Flores-Carreón

Authors
  1. Adolfo López-Esparza
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aurelio Álvarez-Vargas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Héctor M. Mora-Montes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arturo Hernández-Cervantes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ma. Del Carmen Cano-Canchola
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Arturo Flores-Carreón
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Arturo Flores-Carreón.

Additional information

Communicated by Theo Hansen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PPT 386 kb)

Supplementary material 2 (DOC 45 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

López-Esparza, A., Álvarez-Vargas, A., Mora-Montes, H.M. et al. Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity. Arch Microbiol 195, 499–506 (2013). https://doi.org/10.1007/s00203-013-0901-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-013-0901-y

Keywords

Search

Navigation