- Email: [email protected]
N-terminal amino acid sequencing of the 105 kilodalton rhoptry antigen of Plasmodium falciparum
Molecular and Biochemical Parasitology, 33 (1989) 203-204
203
Elsevier MBP 01120 Short Communication
N-terminal amino acid sequencing of the 105 kilodalton rhoptry antigen of Plasmodium falciparum J u a n A . C o o p e r 1, A n n A t k i n s 2 a n d A l l a n J. S a u l 1 ~Queensland Institute of Medical Research, Brisbane, Queensland, Australia and 2Brisbane Centre for Protein and Nucleic Acid Research, Biochemistry Department, University of Queensland, Brisbane, Queensland, Australia
(Received 19 September 1988; accepted 21 November 1988) Key words: Plasmodium falciparum; Rhoptry antigen; Amino acid sequencing
The 140/130/105 kilodalton protein complex is located in the rhoptries of P l a s m o d i u m falciparum merozoites [1]. By peptide mapping, these proteins were found to be unrelated [1]. In some preparations, a 98 kDa and a 38 kDa protein are also found. The 98 kDa protein was shown by peptide mapping to be related to the 105 kDa component. A c D N A clone, Ag44, has been reported which encodes a portion of this antigen [2]. This paper reports further characterization of this 105 kDa component derived from N-terminal amino acid sequencing. The antigen complex from the D10 clone of the FCQ-27/PNG isolate was affinity purified using a monoclonal antibody-Sepharose column, the eluted antigen was electrophoresed on the multizonal electrophoresis (MZE) gel system of Moos et al. [3] and the separated proteins were transferred to polyvinylidene difluoride membrane (Millipore-Waters). Attempts were made to sequence the 140, 130, 105 and 38 kDa proteins directly from the membrane. Neither the 140 kDa nor 130 kDa proteins gave any sequence in several attempts although similar amounts of the 105 kDa and 38 kDa proteins were sequenceable. Both the 105 and 38 kDa protein gave the same N-terminal sequence (Table I) suggesting that the 38 kDa protein, like the 98 kDa protein, is a fragment of the 105 kDa protein. Correspondence address: Juan A. Cooper, Queensland Institute of Medical Research, Bramston Terrace, Herston, Brisbane 4006, Australia.
In an attempt to obtain further sequence data, the 105 kDa protein was electroeluted from a sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel slice and digested with Staphylococcus aureus V8 protease during electrophoresis on an M Z E gel. The relative amount of V8 protease and 105 kDa protein was adjusted to give partial digestion resulting in relatively large fragments [1]. Two of these fragments (68 kDa and 39 kDa) gave the same N-terminal as that sequence derived from the 105 kDa protein but gave a clear signal through substantially more cycles (Table I). The sequence K D V F A G F V T K corresponds to amino acids 25 to 34 deduced from the D N A sequence of the gene coding for this protein (H. Brown et al., manuscript in preparation) and the initial lysine corresponds to a predicted signal peptide cleavage point. We therefore conclude that this is the N-terminal sequence of the mature protein. The previous assignment that a c D N A clone, Ag44, encodes a portion of this antigen [2] relied on the specificity of naturally occurring human antibodies which were affinity purified on the protein expressed by this clone. Because of the frequency of crossreacting antigenic determinants, especially for malarial proteins [4], such assignment must remain tentative. In this case, the amino acid sequence we have determined confirms that Ag44 codes for a fragment of the 105 kDa rhoptry protein.
0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (BiomedicalDivision)
204 TABLE I N-terminal amino acid sequencing of antigens Peptide 105 kDa protein 38 kDa protein 6 8 k D a S . aureusV8peptide 3 9 k D a S . aureusV8peptide
Sequence K K K K
D X X D
V V V V
Initial yield F F F F
A A A A
G X F V T K G F V G F V D/T K
3pmol 3pmol 3pmol 5pmol
X is unknown residue.
Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, by the Genetic Technology Component of the Australian Industry and Development Act 1986 and by UNDP/World Bank/World Health Organization Special Program for Research and
Training in Tropical Diseases. We would like to thank Ms H. Brown from the Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, for providing us with unpublished sequence data.
References 1 Cooper, J.A., Ingram, L.T., Bushell, G.R., Fardoulys, C.A., Stenzel, D., Schofield, L. and Saul, A.J. (1988) The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides. Mol. Biochem. Parasitol. 29,251-260. 2 Coppel, R.L., Bianco, A.E., Culvenor, J.G., Crewther, P.E., Brown, G.V., Anders, R.F. and Kemp, D.J. (1987) A cDNA clone expressing a rhoptry protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 25, 73-81.
3 Moos, M., Nguyen, N.Y. and Liu, T.-Y. (1988) Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem. 263, 6005-6008. 4 Anders, R.F. (1986) Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunol. 8, 52%539.
203
Elsevier MBP 01120 Short Communication
N-terminal amino acid sequencing of the 105 kilodalton rhoptry antigen of Plasmodium falciparum J u a n A . C o o p e r 1, A n n A t k i n s 2 a n d A l l a n J. S a u l 1 ~Queensland Institute of Medical Research, Brisbane, Queensland, Australia and 2Brisbane Centre for Protein and Nucleic Acid Research, Biochemistry Department, University of Queensland, Brisbane, Queensland, Australia
(Received 19 September 1988; accepted 21 November 1988) Key words: Plasmodium falciparum; Rhoptry antigen; Amino acid sequencing
The 140/130/105 kilodalton protein complex is located in the rhoptries of P l a s m o d i u m falciparum merozoites [1]. By peptide mapping, these proteins were found to be unrelated [1]. In some preparations, a 98 kDa and a 38 kDa protein are also found. The 98 kDa protein was shown by peptide mapping to be related to the 105 kDa component. A c D N A clone, Ag44, has been reported which encodes a portion of this antigen [2]. This paper reports further characterization of this 105 kDa component derived from N-terminal amino acid sequencing. The antigen complex from the D10 clone of the FCQ-27/PNG isolate was affinity purified using a monoclonal antibody-Sepharose column, the eluted antigen was electrophoresed on the multizonal electrophoresis (MZE) gel system of Moos et al. [3] and the separated proteins were transferred to polyvinylidene difluoride membrane (Millipore-Waters). Attempts were made to sequence the 140, 130, 105 and 38 kDa proteins directly from the membrane. Neither the 140 kDa nor 130 kDa proteins gave any sequence in several attempts although similar amounts of the 105 kDa and 38 kDa proteins were sequenceable. Both the 105 and 38 kDa protein gave the same N-terminal sequence (Table I) suggesting that the 38 kDa protein, like the 98 kDa protein, is a fragment of the 105 kDa protein. Correspondence address: Juan A. Cooper, Queensland Institute of Medical Research, Bramston Terrace, Herston, Brisbane 4006, Australia.
In an attempt to obtain further sequence data, the 105 kDa protein was electroeluted from a sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel slice and digested with Staphylococcus aureus V8 protease during electrophoresis on an M Z E gel. The relative amount of V8 protease and 105 kDa protein was adjusted to give partial digestion resulting in relatively large fragments [1]. Two of these fragments (68 kDa and 39 kDa) gave the same N-terminal as that sequence derived from the 105 kDa protein but gave a clear signal through substantially more cycles (Table I). The sequence K D V F A G F V T K corresponds to amino acids 25 to 34 deduced from the D N A sequence of the gene coding for this protein (H. Brown et al., manuscript in preparation) and the initial lysine corresponds to a predicted signal peptide cleavage point. We therefore conclude that this is the N-terminal sequence of the mature protein. The previous assignment that a c D N A clone, Ag44, encodes a portion of this antigen [2] relied on the specificity of naturally occurring human antibodies which were affinity purified on the protein expressed by this clone. Because of the frequency of crossreacting antigenic determinants, especially for malarial proteins [4], such assignment must remain tentative. In this case, the amino acid sequence we have determined confirms that Ag44 codes for a fragment of the 105 kDa rhoptry protein.
0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (BiomedicalDivision)
204 TABLE I N-terminal amino acid sequencing of antigens Peptide 105 kDa protein 38 kDa protein 6 8 k D a S . aureusV8peptide 3 9 k D a S . aureusV8peptide
Sequence K K K K
D X X D
V V V V
Initial yield F F F F
A A A A
G X F V T K G F V G F V D/T K
3pmol 3pmol 3pmol 5pmol
X is unknown residue.
Acknowledgements This work was supported by the National Health and Medical Research Council of Australia, by the Genetic Technology Component of the Australian Industry and Development Act 1986 and by UNDP/World Bank/World Health Organization Special Program for Research and
Training in Tropical Diseases. We would like to thank Ms H. Brown from the Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, for providing us with unpublished sequence data.
References 1 Cooper, J.A., Ingram, L.T., Bushell, G.R., Fardoulys, C.A., Stenzel, D., Schofield, L. and Saul, A.J. (1988) The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides. Mol. Biochem. Parasitol. 29,251-260. 2 Coppel, R.L., Bianco, A.E., Culvenor, J.G., Crewther, P.E., Brown, G.V., Anders, R.F. and Kemp, D.J. (1987) A cDNA clone expressing a rhoptry protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 25, 73-81.
3 Moos, M., Nguyen, N.Y. and Liu, T.-Y. (1988) Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem. 263, 6005-6008. 4 Anders, R.F. (1986) Multiple cross-reactivities amongst antigens of Plasmodium falciparum impair the development of protective immunity against malaria. Parasite Immunol. 8, 52%539.