Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells

Yoshifumi Nishikawa, Khajornsak Tragoolpua, Levi H Makala, Xuenan Xuan, Hideyuki Nagasawa

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2.

Original languageEnglish (US)
Pages (from-to)191-201
Number of pages11
JournalVeterinary Parasitology
Volume109
Issue number3-4
DOIs
StatePublished - Nov 11 2002

Fingerprint

Neospora
Glycosylphosphatidylinositols
transmembrane proteins
Neospora caninum
Insects
Baculoviridae
Amino Acids
insects
amino acids
Terminator Codon
stop codon
Tail
Proteins
tail
cells
Phosphoinositide Phospholipase C
neosporosis
phospholipase C
surface antigens
phosphatidylinositols

Keywords

  • Baculovirus
  • GPI
  • Insect cells
  • NcSRS2
  • Neospora caninum
  • Surface protein

ASJC Scopus subject areas

  • Parasitology
  • veterinary(all)

Cite this

Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells. / Nishikawa, Yoshifumi; Tragoolpua, Khajornsak; Makala, Levi H; Xuan, Xuenan; Nagasawa, Hideyuki.

In: Veterinary Parasitology, Vol. 109, No. 3-4, 11.11.2002, p. 191-201.

Research output: Contribution to journalArticle

Nishikawa, Yoshifumi ; Tragoolpua, Khajornsak ; Makala, Levi H ; Xuan, Xuenan ; Nagasawa, Hideyuki. / Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells. In: Veterinary Parasitology. 2002 ; Vol. 109, No. 3-4. pp. 191-201.
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