A novel gene family encoding Maurers cleft transmembrane proteins identified in the subtelomeric reg

1
A novel gene family encoding Maurers cleft
transmembrane proteins identified in the
sub-telomeric regions of Plasmodium falciparum
chromosomes

• Jeffrey R. Johnson, Laurence Florens, Tobili
  Sam-Yellowe, and John R. Yates III

2
Life Cycle of Plasmodium falciparum
Wirth, DF. Nature. 2002 419(6906)495-6
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Anatomy of an Infected Erythrocyte

• A tubovesicular membrane network aids transport
  of secreted proteins to the erythrocyte surface
• Knobs on the surface affect cytoadhesion of
  infected cells

Deitsch, KW and Wellems, TE. 1996 Mol. Biochem.
Parasitol. 76 1-10
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Ultrastructure of Asexual Stages

• Scanning (left) and transmission (right) electron
  micrographs of P. falciparum-infected erythrocytes

Aikawa, et al. 1985 J. Protozool. 32424-429.
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Cytoadhesion
brain (cerebral malaria)

• Employed to sequester infected cells on the
  endothelia of internal organ capillaries
• Circumvents immune detection by preventing
  circulation to the spleen
• Also results in adhesion of uninfected
  erythrocytes (rosetting)

placenta
rosetting
Beeson, et al. 2002 Cell Mol. Life Sci. 59
258-271 Heddini, et al. 2001 Infect. Immun.
69 58495856
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Asexual Stage Characterization

• A panel of antibodies were generated to recognize
  asexual stage specific antigens
• SP1C1 recognizes an integral membrane proteins of
  20 kDa
• SP1A6 recognizes a peripheral membrane protein of
  130 kDa that labels knobs and Maurers clefts
  (right)

Immuno-gold staining with SP1A6 antibody
Sam-Yellow, et al. Parasitol. Res. 2001 87
173-185
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Asexual Stage Characterization

• A panel of antibodies were generated to recognize
  asexual stage specific antigens
• SP1C1 recognizes an integral membrane proteins of
  20 kDa
• SP1A6 recognizes a peripheral membrane protein of
  130 kDa that labels knobs and Maurers clefts
  (right)

Immuno-gold staining with SP1A6 antibody
Sam-Yellow, et al. Parasitol. Res. 2001 87
173-185
8
Asexual Stage Characterization

• A panel of antibodies were generated to recognize
  asexual stage specific antigens
• SP1C1 recognizes an integral membrane proteins of
  20 kDa
• SP1A6 recognizes a peripheral membrane protein of
  130 kDa that labels knobs and Maurers clefts
  (right)

Immuno-gold staining with SP1A6 antibody
Sam-Yellow, et al. Parasitol. Res. 2001 87
173-185
9
Co-localization of SP1C1 and SP1A6
SP1A6
SP1C1

• Confocal immuno-fluorescence microscopy indicate
  co-localization
• SP1C1 target is indirectly localized to knobs and
  Maurers clefts
• Direct IEM using SP1C1 was unsuccessful

DAPI
Overlay
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Multidimensional Protein Analysis
Protease Digestion
P. falciparum immuno-precipitated samples
SCX
RP
microcapillary column
SEQUEST software
MS/MS datasets
genomic databases
Ion-trap tandem mass spectrometer
locus peptide
------------------------- ------------------------
----------- PF01002
DDSATGFAGDLNDDCKNAHIFKA PF01002
EFLGLLNNETTCTKKLND PF01002
AMVCALTYEEKTSSASGGEKNTT PF01110
KGNSKSNFIPFRD PF01110
KNISKVCQNKSTINVPKV PF01110
ESGAIINNNNDEQHNNV PF01118
KQYEQIRQNEKI
Summarizing software, DTASelect and Contrast
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• Peptides were detected from 10 highly conserved
  proteins (colored shading)
• 5 proteins had two exons, 5 proteins had only one
  exon
• All proteins had two predicted transmembrane
  domains separated by a short stretch of amino
  acids

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Genome Sequence Analysis

• Inspection of raw DNA sequences revealed a two
  exon structure for all proteins in the family
• Errors in gene prediction common in P. falciparum
• Methods to correct gene modeling errors using
  peptides detected in predicted non-coding regions
  are possible

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• Peptides were detected from 10 highly conserved
  proteins (colored shading)
• 5 proteins had two exons, 5 proteins had only one
  exon
• All proteins had two predicted transmembrane
  domains separated by a short stretch of amino
  acids

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• Peptides were detected from 10 highly conserved
  proteins (colored shading)
• 5 proteins had two exons, 5 proteins had only one
  exon
• All proteins had two predicted transmembrane
  domains separated by a short stretch of amino
  acids

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Confirmation with Microarray Data

• The Affymetrix chip includes probes within
  non-coding regions
• Probes in the corrected N-terminal region verify
  gene-modeling errors
• 7 of 11 genes show expression during the
  trophozoite stage
• 3 genes lacking expression do not include more
  than one unique probe
• MAL7P1.58 had 11 probes covering its sequence
• None indicate expression

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An Exposed Polymorphic Region
PFB0985c ..NLWNNIWIVSTTLLLIIIMI--ACIIVCTPETYTALYP
AFILLIFIIHIVARYFPDM.. PFA0680c ..NLWNNIWIVSTTLLLI
IIMI--VCLFSVDITSSSALGPAFILLIFIIHIVARYFPDM.. PFC108
0c ..NLWNNIWINSITLTLIIIMI--ACVFSVEITSSSALYPAFILLI
FIIYIYARYFPEM.. PF11_0014 ..NFWNNIWINSINFFVIIIMI--
ACVVVGLIEASSISYPLCIFPVFMLYMLVRFFPDM.. MAL7P1.5 ..
NLWNNIWINYITLSLIIIMIALLFLLPGS-QPYYIFYSAFSLLIFIIYIV
ASYFPDM.. PF11_0025 ..NLWNNIGINSITLSLIIIMIMIACMFAI
CNAPQYIYVPASFLLIFIIYIVARYFPDM.. MAL6P1.15 ..NLWNN
IGINSITLSLIIIMIMIACMFAIYSTAQYIYVPASFLLIFIIYIVARYFP
DM.. PF10_0390 ..NLWNNIWIVSTTLSLIIICIAFVFYLASTHVS
SQLYYFAFSPLIFIIYIVARYFPDM.. PFA0065w ..NLWNNIWIVS
TTLLLIIIMIACVYWLASTPPPG-LFCLPFIVLIFIIYIVARYFPDM..
PFB0960c ..NTWNNIWIVTMTLFLIIIMI--ACIFSVGITHASAFYP
ALFLAVFLIYMYARFFPKI.. MAL7P1.58 ..NFWNNIWIVVITLLSI
ILLI--AAMAMNTFPGFSAFLVTFVLSNLMIYMFARFYPEI..
. .
. .

• The only unconserved region overlaps with an
  extracellular loop
• Similar structure for PfEMP1, rif, and stevor

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Chromosome Locations

• 10 of 11 family members found in sub-telomeric
  regions of chromosomes

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Conclusions Future Directions

• Combining immunoprecipitation with MudPIT, we
  have identified a novel sub-telomeric gene family
  encoding proteins localized to the erythrocyte
  surface and Maurers clefts
• Expression of nearly all genes during the
  trophozoite stage confirmed at the transcript
  level
• In silico predictions indicate potentially
  phosphorylated residues
• Some modified peptides have been identified with
  low confidence
• Digestion with diverse proteases yielding
  similarly modified peptides increase confidence

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Acknowledgements

• Laurence Florens, The Stowers Institute
• Tobili Sam-Yellowe, Cleveland State Univ.
• Daniel Carucci, Naval Medical Reseach Center
• Karine Le Roch Elizabeth Winzeler, Insitute for
  Childhood and Neglected Diseases
• This work supported by the Office of Naval
  Research, the US Army Medical Research and
  Material Command, and the National Institutes of
  Health
• JRJ wishes to acknowledge the LJIS
  Interdisciplinary Training Program and The
  Burroughs-Wellcome Fund for fellowship support

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Database Queries

• BLAST queries revealed MAL7P1.58
• No significant results from non-redundant BLAST
  query

• Instead P. falciparum and P. yoelii databases
  were searched for proteins by structural features
• 200-300 amino acids in length
• Predicted N-terminal signal peptide sequence
• Consecutive transmembrane domains

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Acknowledgements

• Laurence Florens, The Stowers Institute
• Tobili Sam-Yellowe, Cleveland State University
• Karine Le Roch Elizabeth Winzeler, Dept. of
  Cell Biology
• JRJ wishes to acknowledge the LJIS
  Interdisciplinary Training Program and The
  Burroughs-Wellcome Fund for fellowship support

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