The secretome analysis of Tetrahymena thermophilia under different growth conditions.

1. Effects of Strain Type and Growth Conditions on the Secretome of Tetrahymena thermophila
CL K CH JS Madinger1; Collins2; Taron1; Benner1
1New England Biolabs Inc., Ipswich, MA; 2University of California Berkeley, Berkeley, CA
Overview
Rich Media Starvation A.
• Three strains of Tetrahymena thermophila (Tt1, Tt2 and Tt3) were cultured under
two conditions (rich media and starvation) and secreted proteins were identified by M Tt1 Tt3 Tt2 Std. M Tt1 Tt3 Tt2 Rich Media Secretome
online ESI-MS/MS. kDa
• Growth conditions affect the protein composition of the T. thermophila secretome. 212
Proteins in common between different conditions are called the “base secretome”. Protease
• Additionally, whole cell proteins of Tt2 were identified by 2D-LC MS/MS. Tt1
116 Tt2 Secretory Transport
• There were 43 proteins in common between the Tetrahymena thermophila Tt3
secretomes (6 conditions) and whole cell extract. Signaling
66 Figure 7. Distribution of the detected T. thermophila
Unknown secretome proteins according to their different GO
Introduction terms: A) Tt1 (outer circle), Tt2 (middle circle) and Tt3
43 Structural
Tetrahymena thermophila, a ciliated protozoa, is inexpensive to culture and can be (inner circle) cultured under starvation conditions; B)
grown to high cell densities. While T. thermophila appears to be an excellent B. Protein Synthesis and Folding Tt2 cultured under rich media (outer circle) and
candidate for use in heterologous protein expression, it is essential to know what Glycolytic Enzyme starvation (inner circle)
27
proteins are intrinsically secreted. Some proteins within the secretome may be
detrimental to heterologous protein expression. Detrimental proteins of T. Glycosyl Hydrolase
thermophila could be genetically modified to suppress their expression and therefore Stress Response
optimize protein expression. It is possible that some detrimental protein expression 14
Defense
can be controlled by strain type and/or growth condition, therefore rendering genetic
manipulation unnecessary. Identification of proteins intrinsic to its secretome is the Other
Figure 5. Venn diagram showing the intersection of the proteins secreted by different
first step in the optimization of T. thermophila for its use in heterologous secreted Figure 2. Analysis of the supernatants of different T. thermophila strains (Tt1, Tt2 and strains of T. thermophila (Tt1, Tt2 and Tt3) when cultured with rich media and
protein expression. Tt3) cultured under two conditions (rich media and starvation) by sodium dodecyl sulfate detected by mass spectrometry. Data is highlighted further in Table 2.
polyacrylamide gel electrophoresis (SDS-PAGE). Lane 5 (Std.) is New England Biolabs
Methods protein marker, the bands corresponding to 27 and 66 kDa are most intense. Lanes 1
Strains and Culture Conditions through 4 contain samples grown with rich media: Lane 1 is media (M), Lanes 2, 3 and 4 Table 2. T. thermophila base secretome proteins identified when cultured with rich media
Tetrahymena thermophila (Figure 1) strains Tt1, Tt2 and Tt3 were cultured in flasks are strains Tt1, Tt3 and Tt2, respectively. Lanes 6 through 9 contain samples that were
TTHERM_00066890 Peptidase C13 family protein
at 30°C with 150 rpm shaking. Cells were cultured in Neff media (0.25% proteose starved: Lane 6 is media (M), Lanes 7, 8 and 9 are strains Tt1, Tt3 and Tt2, respectively. Results
TTHERM_00079450 Mitochondrial glycoprotein
peptone, 0.25% yeast extract, 0.5% glucose, 3.3 μM FeCl3) and the samples were TTHERM_00079640 Papain family cysteine protease • In total, 492 proteins were identified from the analysis of Tetrahymena thermophila cultured under multiple conditions and highly expressed in
Papain family cysteine protease
divided when cells were in log phase of population growth (~2x105/mL). Half of the TTHERM_00079650 whole cells. There are a total of 24,725 proteins in the predicted proteome [2].
TTHERM_00086780 CCF12; C-terminal crystallin fold protein, dense core granules
culture continued growing overnight into stationary phase (~1x106/mL) while the Hypothetical
• A secreted compliment of 247 proteins were identified from the analysis of Tetrahymena thermophila cultured under six different conditions:
TTHERM_00094060
other half was harvested and transferred to starvation media (10 mM Tris-HCl, pH TTHERM_00161130 Papain family cysteine protease strains Tt1, Tt2 and Tt3 cultured under both rich media and starvation conditions.
7.5) for starvation overnight. TTHERM_00268060 Papain family cysteine protease/CysP5 • We identified 288 proteins from our analysis of T. thermophila whole cells, which is similar to the number previously reported (223) for cilia
For the protein secretion analysis, cells were harvested in a tabletop Eppendorf TTHERM_00382240 Hypothetical proteins [3]. Of the 288 proteins identified, only 43 total proteins were detected in the combined secretomes of T. thermophila cultured under
Hypothetical; chitinase active site glycoside hydrolase domain six different conditions (data not shown). Considering strain-specific results, only 1 protein was in common between the whole cell proteins
centrifuge at no more than 4000 rpm. The spent culture media was filtered (acrodisc TTHERM_00442170
TTHERM_00515220 Hypothetical; chitinase active site glycoside hydrolase domain
0.2 micron low protein retention syringe filter) and concentrated (20 to 50-fold) by Papain family cysteine protease/CysP1
and secreted proteins from Tt1 cultured under rich media and starvation conditions (Figure 3).
TTHERM_00530660
Vivaspin centrifugation (10 kDa cut-off). These samples were then aliquotted for Hypothetical • Far fewer proteins were secreted by T. thermophila strains Tt1, Tt2 and Tt3 when cultured under starvation conditions versus rich media
TTHERM_00566690
analysis by SDS-PAGE and MS. TTHERM_00590090 CMB1/p85; calmodulin-binding protein; roles in cytokinesis and phagocytosis (Figures 4 & 5). Additionally, the base secretome of T. thermophila strains Tt1, Tt2 and Tt3 was much smaller when cultured under starvation
For the whole cell analysis, cells from T. thermophila strain Tt2 were pelleted from TTHERM_00606960 SerH3 immobilization antigen; GPI-linked cell surface antigen conditions (11 proteins; Figure 4) than when cultured with rich media (33 proteins; Figure 5).
overnight growth in Neff media and frozen at -80°C. The pellet was resuspended in TTHERM_00641150 Papain family cysteine protease; microarray 11th top constitutive • There is little overlap in the secreted proteins detected between starvation and rich media conditions for the T. thermophila strains Tt1, Tt2, and
TTHERM_00660360 Papain family cysteine protease
50 mM Tris, pH 8, 0.1% SDS and boiled for 3 minutes at 97°C. The sample was Tt3 (Figures 6A, B, and C, respectively). For instance, 40 secreted proteins were detected by MS when strain Tt3 was cultured under starvation
TTHERM_00660380 Papain family cysteine protease
then brought to 0.1% TFA and spun to remove cellular debris. Papain family cysteine protease conditions whereas 65 proteins were detected with rich media: of these proteins, only 17 were detected under both conditions (Figure 6C).
TTHERM_00660390
TTHERM_00661740 Hypothetical; one of familyA of closely related hypothetical proteins
Multi-dimensional Protein/Peptide Separation TTHERM_00662750 Hypothetical; one of familyA of closely related hypothetical proteins
Proteins from the T. thermophila whole cell sample and T. thermophila Tt1, Tt2 and TTHERM_00663790 Hypothetical; one of familyA of closely related hypothetical proteins
PLA1/CysP3; Lysosomal phospholipase A1; "may be secreted into the environment as part
Tt3 samples grown under vegetative conditions were separated by HP-RPLC via a of an attack and defense system,"
TTHERM_00683010 Discussion
PLRP-S column. Fractions were collected, dried to completion, resuspended in Papain family cysteine protease/CysP2
TTHERM_00683060 • Since there was minimal overlap between the secreted and whole cell proteins detected (Figure 3), we conclude that cell lysis is a negligible
reaction buffer and digested overnight with TPCK-treated Trypsin (New England TTHERM_00755950 Papain family cysteine protease/CysP4
contribution to proteins detected in the “secretome” of Tt1, Tt2 and Tt3.
Biolabs). Peptides were then separated by an integrated C18 trap/column/needle and TTHERM_00760310 Papain family cysteine protease/tetrain/CysP6
Papain family cysteine protease/CYP1; starvation-induced cysteine protease • Growth condition (rich media versus starvation) affected T. thermophila protein expression both quantitatively (Figure 2) and qualitatively
analyzed online by nanoESI-MS/MS with an Ion Trap Mass Spectrometer (Agilent TTHERM_00881440
Figure 3. Venn diagram showing the intersection of the proteins secreted by TTHERM_00881450 Papain family cysteine protease (Figure 7). For instance, the functional groupings and the populations of these groups were both affected when strain Tt2 was cultured under
Technologies) and ChipCube. These analyses were performed in triplicate.
T. thermophila strain Tt1 whole cell and strain Tt1 secreted proteins cultured with TTHERM_00951910 Hypothetical; Glycoside hydrolase, chitinase active site domain rich media and starvation conditions (Figure 7B). This variance was also observed when comparing the three strains cultured under the
TTHERM_00951920 Hypothetical; Glycoside hydrolase, chitinase active site domain starvation condition (Figure 7A). Therefore we cannot say whether variance in strain or culture condition had a greater qualitative effect on
Single-dimensional Peptide Separation rich media and starvation conditions. Hypothetical
TTHERM_01043150 protein expression.
Proteins from the T. thermophila Tt1, Tt2 and Tt3 strains grown under starvation TTHERM_00662749
• Regardless of strain or culture conditions, proteases always comprise the largest group of proteins identified to be expressed (<25%, Figure 7).
conditions were dried to completion, resuspended in reaction buffer and digested (373600685) Hypothetical
TTHERM_00102779 cathepsin z; 70% similarity to TTHERM_00102770 Papain family cysteine protease Since this was not a quantitative study, we can not presently determine whether just a broad spectrum of proteases are secreted or what the
overnight with TPCK-treated Trypsin (New England Biolabs). Peptides were
(704849) containing protein variation in the levels of secreted proteases are. Due to the high number of proteases expressed under all culture conditions and in all strains no
separated by an integrated C18 trap/column/needle and analyzed online by nanoESI-
one condition examined may be ideal for general protein expression.
MS/MS with an Ion Trap Mass Spectrometer (Agilent Technologies) and ChipCube.
These analyses were performed in triplicate.
Starved Media Secretome • The consistently second largest functional grouping of secreted proteins remain functionally uncharacterized (Figure 7). The elucidation of the
function of the uncharacterized proteins of T. thermophila is important for a full interpretation of our results.
• T. thermophila grown under certain conditions secrete a mucus or slime. This mucus makes direct shotgun MS/MS analysis difficult or
Protein Identification using MS and MS/MS Data
impossible. Our 2D-LC methodology separates the mucus from the protein and no further interference with MS/MS data collection occurs.
The MS/MS data were analyzed using Mascot (Matrix Science) and Spectrum Mill A.
(Agilent Technologies). Peptides generated by a tryptic digest were searched against
the T. thermophila genome in a T. thermophila database that was generated by
combining the T. thermophila genome with Swiss-Prot database (Version 51.6).
Proteins scoring greater than 67 and 20 were considered valid identifications (for Conclusions
Mascot and Spectrum Mill, respectively) and were combined to generate the final The Tetrahymena thermophila secretomes from multiple strains and culture conditions were examined by both 1D- and 2D-LC MS/MS
list of identified proteins. analyses. Additionally, we used 2D-LC MS/MS to examine the proteins expressed at high levels in whole cells of Tetrahymena thermophila.
We have had success using this method in previous secretome analyses [4,5] and the 2D separation was essential for our analyses here because
of the additional complexity present with Tetrahymena samples.
B. The overlap observed between the secretome and whole cell proteins suggests that the proteins detected are a result of secretion rather than
cell lysis. In examining the individual data sets, there were several unexpected results. First, more proteins characterized as being part of
protein synthesis appear to be expressed during starvation conditions. Second, the percentage of proteases detected stays relatively constant
regardless of strain or growth condition. And finally, the high number of unknown proteins identified indicates that further elucidation is
required before our results can be completely interpreted.
Figure 4. Venn diagram showing the intersection of the proteins secreted by
different strains of T. thermophila (Tt1, Tt2 and Tt3) when cultured under starvation
conditions and detected by mass spectrometry. Data is highlighted further in Table 1.
References
Table 1. T. thermophila base secretome proteins identified when cultured under C. 1. Image courtesy of Brady Culver, University of Colorado.
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