Download Functional Conservation of Calreticulin in Euglena gracilis

Functional Conservation of
Calreticulin in Euglena gracilis
Kym Craft
Rick Mohanty
05 October 2005
What is calreticulin?
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High capacity, low affinity Ca2+ binding protein
Can hold 20 moles of Ca2+
Molecular Mass of 50-60 kDa
Localized in ER of eukaryotes (eg. E. Hux)
Integral to signal transduction pathways
involving Ca2+ as second messenger
Why in the ER?
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Calreticulin is characteristically found in the ER
Possesses short signal peptide
Possesses KDEL retention signal
Ideal location for signal transduction pathways
Converting one signal/stimulus into another
 Influences how cell can react and respond to
environment.
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Euglena gracilis
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Unicellular eukaryotic protist
Branched off relatively early in eukaryotic
evolution
Plastids have three outer membranes instead of
two.
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Suggests engulfment of eukaryote, which partially
integrated into cell structure.
Kinetoplastid
Chlorophyte
Current Euglena
• Kinetoplastid is an ancestor of Euglena
•Kinetoplastid engulfs chlorophyte, and incorporates some of its DNA,
also incorporates chloroplast
• Did calreticulin come from symbiont or host? Was the symbiont a
chlorophyte at all?
Questions Posed
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Is the Euglena protein calreticulin?
How far back can components of Ca2+ homeostasis
be traced within contemporary eukaryotes?
What is the evolutionary order of appearance of
calreticulin?
If a host eukaryote engulfed a photosynthetic
eukaryote, would components of the symbiont be
incorporated into the host’s Ca2+ machinery?
Cell Disruption
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Euglena cells were grown, then disrupted by:
French Press: 10,000 psi on target cells
 Acid-Washed Glass Beads: After cells were initially
broken, organelles were kept intact by this method.
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Protein Purification
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Acidic Ca2+ proteins were isolated by
Ammonium Sulphate Precipitation.
Followed by DEAE-Cellulose chromotography
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Separation technique based on ion exchange
Cell Fractionation
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Organelles separated by Sucrose Density
Gradient Centrifugation at 100,000g
Organelles sediment in layer that matches their
own density.
Biochemical Techniques Electrophoresis
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Two Dimensional Electrophoresis
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Separates by molecular mass and pH (4-6.5)
SDS-PAGE - Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
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Separates proteins, and can estimate molecular mass
Biochemical Techniques –
Immunoblot Analysis
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Blots were incubated with:
Antibodies against rabbit calreticulin
 Antibodies against spinach calreticulin
 Cross-Reacted with spinach, no reaction with rabbit
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1.
Euglena in spinach
antiserum
2.
Spinach in spinach
antiserum
Molecular Techniques
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Amplification of calreticulin probe: PCR of
degenerate nucleotides
Probe: Detailed piece of DNA, chemically labeled
and used to locate sequences.
 Redundancy in genetic code, multiple codons.
Contain different triplets, yet code same amino acid
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The product was 260bp, and it was amplified
again
Molecular Techniques
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This product was purified, then transformed
into E. coli yielding the plasmid pPCRcalrH
A specific fragment Cla I-XhoI, was isolated by
electrophoresis, then sequenced.
BLAST searches and ClustalW were used to aid
in sequence analysis
Electrophoresis Analysis
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Lane 1 shows a 56kDa
protein
Lane 2 shows a 56kDa
protein that can readily
bind Ca2+
Lane 3 shows 56kDa
protein with acidic
character
All characterisic of
calreticulin
Positively Calreticulin
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All known calreticulins possess sequence
DCGGGY
Sequence analysis of Euglena cDNA yielded 5
matches for calreticulin
Calreticulin is known not to phosphorylate
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Euglena did not phosphorylate by protein kinase
CK2
Protein Localized in ER
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Sucrose Density
Centrifugation showed
results indicative of ER
• cDNA analysis showed
calreticulin signal peptide
in N-Terminus
•Also showed KDEL retention signal in C-terminus
•Indicative of calreticulin, and localization in the ER
Symbiont or Host?
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Previous studies have shown that Euglena arose
through secondary symbiosis.
Euglena showed genes of kinetoplastid and
chlorophyte
Phylogenetic analysis shows Euglena calreticulin
branches off from the Leishmania donovani, a
kinetoplastoid.
The Ca2+ apparatus derived from the host not the
symbiont.
cDNA analysis (KDEL, signal peptide), and
phylogenetic analysis suggest that the Ca2+ mechanism
was already in place at the time of divergence.
Calreticulin Phylogenetic Tree