The effects of sugars on the secretome of Kluyveromyces lactis

1. The nature of glycosylation in the yeast Kluyveromyces lactis
CL Swaim; ML Cushing; L Fields; J Canovas; S Sharma; CH Taron; JS Benner
New England Biolabs Inc., Ipswich, MA 01938
Glycosylated K. lactis secretome proteins detected by MS without PNGase F treatment
Overview
• Different carbon sources (glucose, galactose, glycerol) affect the protein Example MS/MS spectrum from Agilent 6330 Ion-Tap with ChipESI
composition of the Kluyveromyces lactis (K. lactis) secretome. 1 23456 78 1 2 3 4 5 6 7 8 1 2 3 4 5 6 78
• Glycosylated proteins in the secretome of K. lactis were analyzed by 2D-LC MS A. B. C.
/MS:
– N-linked mannosyl-core glycoproteins were affinity purified using a
Concavalin A-Sepharose 4B 66- 66- 66-
– Proteins were digested using Trypsin
– Resulting peptides were separated by reversed phase (C18 column) and 31- 31- 31-
analyzed via online ESI-MS/MS using an integrated column and nanospray 14-
needle (ChipESI). 14- 14-
– Protein identification was conducted with Spectrum Mill and Mascot
• Experimentally detected glycosylated secretome proteins from K. lactis grown with
different carbon sources were compared to each other and to proteins predicted in
silico to comprise the secretome.
Figure 4. Separation by SDS-PAGE of the K. lactis supernatants after growth with different carbon sources
(A. glucose; B. galactose; C. glycerol) and purification with lectin affinity chromatography (ConA). Samples
Figure 1. Picture of K. lactis (10 L) were mixed with 3 L Blue Loading Buffer, the entire volume was added to the different sample Figure 2. Representative data from the galactose data set.
Introduction (http://www.atcc.org) wells and the gel was run at 115 V. The gel was then silver stained (Owl Separation Systems). Lanes 1 and 2
Glycosylation of proteins is the most abundant of post translational modifications in correspond to protein standards (BioLabs and Novex, respectively) with their corresponding molecular
all eukaryotic organisms. Previously, we examined the secretome of the yeast masses indicated in kDa on the left. Lane 3 corresponds to the sample that was loaded onto the ConA column
Figure 5. Venn diagram showing the intersection of the
Kluyveromyces lactis (K. lactis) and how the protein composition of this secretome (“Load”) and lane 4 corresponds to the sample that was not retained by the ConA column (“Flowthrough”).
proteins secreted by K. lactis when grown in different
is directly affected by adjusting the fermentation conditions [1]. The goal of this Lanes 5 and 6 contain the sample that resulted from washing the ConA column with the Column Buffer Results
media (glucose, galactose, glycerol), purified by
work is to observe how changing the carbon source provided to K. lactis during 1 2 3 4 5 6 7 8 9 10 (“Wash 1” and “Wash 2”, respectively; see Materials and Methods). Samples that eluted with the addition of • PNGase F treatment removes N-linked carbohydrates and indicates that K. lactis secretes many
affinity chromatography and detected by mass
fermentation affects the secretion of glycosylated proteins. the elution buffer (0.5M methyl mannose) are separated in lanes 7 and 8 (“Elutant 1” and “Elutant 2”, N-linked glycosylated proteins (Figure 3)
spectrometry. Data is color coded to match the results
respectively). • In general, carbon source does not affect the K. lactis secretome proteins that are glycosylated
in Table 1.
(Figure 5, Table 1). The greatest number of glycosylated proteins was detected when glucose was
used as the carbon source: 13. There were 12 and 9 glycosylated protein detected when galactose
and glycerol were used as the carbon sources, respectively. Of these proteins, 9 were common to
Methods 66- Table 1. Proteins identified from the analysis of the K. lactis secretome after lectin affinity purification.
all three sugar sources.
Strains and Fed-batch Fermentation • Treatment with PNGase F only cleaves the N-glycan bond. Several proteins from the glucose,
K. lactis Open S. cerevisiae Open S. cerevisiae detected in detected in detected in predicted
Kluyveromyces lactis (Figure 1) strain GG799 (New England BioLabs Inc.) was 31- Reading Frame Reading Frame gene name glucose galactose glycerol in silico function glycerol and galactose carbon sources remained glycosylated after treatment with PNGase F
used in all fermentations. Fermentations were conducted in a 2 L Bioflo 110 (Figure 6), suggesting that these proteins may be modified by O-glycosylation.
KLLA0A03201g YMR305C SCW10 X X X X putative glycosyl hydrolase - hydrolyzing O-glycosyl compounds
fermenter (New Brunswick Scientific). The batch medium contained various salts,
metals and cofactors with 20 g L-1 of Glucose for all fermentations. Pre-cultures
14- KLLA0B06347g YKL096W CWP1 X X X Cell wall mannoprotein • Of the proteins identified after PNGase F treatment (Figure 7, Table 2), only KLLA0A03201g
KLLA0B07370g YKL164C PIR1 X X X X O-glycosylated protein required for cell wall stability and KLLA0B06347g have Serine-rich regions in their primary sequence, a characteristic of O
were grown at 30°C and used to inoculate 0.8 L of batch medium (2.5% v/v) in the KLLA0B07392g YKL164C PIR2 X X X X O-glycosylated protein required for cell wall stability -glycosylated proteins. The sequences of several other glycosylated proteins also had regions rich
fermenter. The pH and temperature during the fermentations were maintained at 6 KLLA0B09746g YBR162C TOS1 X X X Covalently-bound cell wall protein of unknown function in Serine (KLLA0B09746g, KLLA0C14047g, KLLA0F04433g, KLLA0E10703g and
and 30°C, respectively. Air was sparged into the fermenter at a constant rate. The KLLA0C14047g YGR279C SCW4 X X X X Cell wall protein with similarity to glucanases KLLA0E14982g), but were not identified after PNGase F treatment.
dissolved oxygen (DO) was maintained at 30% of saturation by varying the KLLA0C14454g YBR078W ECM33 X X GPI-anchored protein of unknown function, has a possible role in apical bud growth • The majority of the glycosylated proteins detected were predicted in silico to be GSP-secreted
agitation rate and supplementing the inlet air with pure oxygen as necessary. The KLLA0F04433g YNL066W SUN4 X X X X Protein of the SUN family
proteins (Tables 1&2).
batch phase of the fermentation was approximately 15 hours, at which time the Figure 3. Analysis of the K. lactis supernatants after growth KLLA0A11748g no homolog no homolog X X unknown
glucose in the batch medium was completely consumed. Feeding was initiated at an with different carbon sources by sodium dodecyl sulfate KLLA0B07447g YJL158C CIS3 X X X X Mannose-containing glycoprotein constituent of the cell wall
exponential rate to control the growth rate at approximately 0.1 hr-1 and obtain a polyacrylamide gel electrophoresis (SDS-PAGE) using a KLLA0E10703g YGL028C SCW11 X X X unk Cell wall protein with similarity to glucanases
10-20% polyacrylamide gradient Tris-Tricine buffer gel. KLLA0E14982g YKR042W UTH1 X X X X SUN family member required for mitochondrial autophagy
high cell concentration [2,3]. The feed medium contained 448 g L-1 of Glucose,
KLLA0F05753g YDL178W DLD2 X unk D-lactate dehydrogenase
Galactose or Glycerol as the carbon source with various salts, metals and cofactors. Samples (10 L) were mixed with 3 L Blue Loading
KLLA0F11704g YNR067C DSE4 X unk daughter cell-specific secreted protein with similarity to glucanases Conclusions
The final cell density was 79.8 g L-1, 66.9 g L-1 and 25.92 g L-1 dry cell weight for Buffer, the entire volume was added to the different sample
Glucose, Galactose and Glycerol feeds respectively. Yeast cells were removed by wells and the gel was run at 115 V. The gel was then silver The K. lactis secretome from multiple carbon sources was examined using a 2D-LC MS/MS
centrifugation at 20,000 x g for one hour. The supernatants from these three sugar stained (Owl Separation Systems). Lanes 1 and 2 approach. The secretome from these carbon sources was comprised of 158 proteins (Figure 8).
samples were used in all further experiments. correspond to protein standards (BioLabs and Novex, Only a limited subset of the proteins in the secretome (14 of 158) were found to contain a
respectively) with their corresponding molecular masses mannose rich core. The detection of protein purified by lectin affinity chromatography after the
Lectin Affinity Chromatography and online ESI-MS/MS Analysis
The different concentrated yeast stock solutions were added (50 L) to a Concavalin
indicated in kDa on the left. Lanes 3 and 4 were the Rnase B
standard in the presence and absence of PNGase F,
Glycosylated K. lactis secretome proteins detected by MS with PNGase F treatment samples were treated with PNGase F indicates that either some K. lactis secretion proteins are
modified by O-glycosylations or that some N-glycosylations are inaccessible to PNGase F. Three
A-Spharose 4B (ConA) column (GE Healthcare) and washed with column buffer respectively. The K. lactis supernatant with glucose as the of these glycoproteins were found to still be retained by a ConA affinity chromatography after
(10 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1 mM CaCl2, 1 mM MnCl2). Glycosylated sugar source in the presence and absence of PNGase F was PNGase F treatment from all the carbon sources and an additional two proteins from growth on
proteins were eluted using 0.5 M methyl mannose. Collected fractions were diluted analyzed in lanes 5 and 6, respectively. The supernatant with 1 23 4 56 78 1 2 3 4 5 6 7 8 1 2 3 4 5 6 78 glucose. It is likely that these are O-glycosylations since annotations of the corresponding S.
in Trypsin buffer and digested with Trypsin (New England BioLabs, Inc.) overnight galactose was analyzed in lanes 7 and 8 (in the presence and A. B. C. cerervisiae proteins are to O-linked-glycoproteins.
(at 37 °C) at a 1:20 enzyme to protein ratio. Resulting peptides were washed with absence of PNGase F, respectively) and the supernatant with
C18 Zip Tips (Millipore), eluted with 30% ACN and dried to completion. Peptides glycerol was analyzed in lanes 9 and 10 (in the presence and
were separated by an integrated C18 trap/column/needle and analyzed online by absence of PNGase F, respectively). 66- 66- 66-
nanoESI-MS/MS with an Ion Trap Mass Spectrometer (Agilent Technologies) and
ChipCube. Representative data is shown in Figure 2. PNGase F treatment was 31- 31-
performed using glycerol-free enzyme (New England BioLabs, Inc.).
31-
14- References
14-
14- 1. Swaim CL, Anton BP, Sharma SS, Taron CH, Benner JS. (2008) Proteomics, DOI:10.1002
Protein Identification using MS and MS/MS Data /pmic.200700764.
The MS/MS data were analyzed using Mascot (Matrix Science) and Spectrum Mill 2. Akesson M, Karlsson E, Hagander P, Axelsson J, Tocaj A. (1999) Biotechnology and
(Agilent Technologies). Peptides generated by a tryptic digest were searched against Bioengineering 64(5):590-598.
the K. lacis genome in a K. lactis database. Proteins scoring greater than 67 and 20 3. DeLisa MP, Li J, Rao G, Weigand WA, Bentley WE. (1999) Biotechnology and
Figure 6. Separation by SDS-PAGE of the K. lactis supernatants after growth with different carbon sources (A.
were considered valid identifications for Mascot and Spectrum Mill, respectively. Bioengineering 65(1):54-64.
glucose; B. galactose; C. glycerol), treatment with PNGase F and purification with lectin affinity
Proteins scoring 60 in Mascot were also considered if they were simultaneously 4. Lee SA, Wormsley S, Kamoun S, Lee AF, Joiner K, et al. (2003) Yeast 20: 595-610.
chromatography (ConA). Samples (10 L) were mixed with 3 L Blue Loading Buffer, the entire volume was
identified by Spectrum Mill. 5. Sonnhammer EL, von Heijne G, Krogh A (1998) Proc Int Conf Intell Syst Mol Biol 6:
added to the different sample wells and the gel was run at 115 V. The gel was then silver stained (Owl
Separation Systems). Lanes 1 and 2 correspond to protein standards (BioLabs and Novex, respectively) with 175-182.
Computational prediction of the K. lactis secretome Figure 7. Venn diagram showing the intersection of the 6. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) J Mol Biol 305: 567-580.
their corresponding molecular masses indicated in kDa on the left. Lane 3 corresponds to the sample that was
The in silico analysis was conducted using the method of Lee and coworkers [4] proteins secreted by K. lactis when grown in different 7. Eisenhaber F, Eisenhaber B, Kubina W, Maurer-Stroh S, Neuberger G, et al. (2003) Nucleic
loaded onto the ConA column (“Load”) and lane 4 corresponds to the sample that was not retained by the ConA
with minor modifications as follows. Only proteins with an N-terminal general media (glucose, galactose, glycerol), treated with Acids Res 31: 3631-3634.
column (“Flowthrough”). Lanes 5 and 6 contain the sample that resulted from washing the ConA column with
secretory pathway (GSP) sequence were considered as part of the K. lactis PNGase F, purified by affinity chromatography and 8. Eisenhaber B, Bork P, Eisenhaber F (1999) J Mol Biol 292: 741-758.
the Column Buffer (“Wash 1” and “Wash 2”, respectively; see Materials and Methods). Samples that eluted
secretome. This sequence was predicted using SignalP 3.0, and a positive SignalP detected by mass spectrometry. Data is color coded to 9. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) J Mol Biol 300: 1005-1016.
with the addition of the elution buffer (0.5M methyl mannose) are separated in lanes 7 and 8 (“Elutant 1” and
hit was defined as having a signal peptide predicted by both the SignalP-Neural match the results in Table 2.
“Elutant 2”, respectively).
Network (NN) and SignalP-Hidden Markov Model (HMM) algorithms and where
the predicted signal peptide cleavage site was between residues 10 and 40 from the
N-terminus. There were several parameters that excluded proteins from the Table 2. Proteins identified from the analysis of the K. lactis secretome after treatment with PNGase F and lectin affinity purification.
secretome. First, proteins possessing a transmembrane domain were excluded
unless the domain was found within the first 40 N-terminal residues. K. lactis Open S. cerevisiae Open S. cerevisiae detected in detected in detected in predicted
Transmembrane domains were predicted using TMHMM 2.0 [5,6]. Second, Figure 8. Venn diagram showing the intersection of the Reading Frame Reading Frame gene name glucose galactose glycerol in silico function Acknowledgments
proteins containing a glycosylphosphatidylinisotol (GPI) anchor were excluded. secretome proteins of by K. lactis when grown in KLLA0A03201g YMR305C SCW10 X X putative glycosyl hydrolase - hydrolyzing O-glycosyl compounds The authors would like to thank New England Biolabs and Don Comb for their support. We also
KLLA0B06347g YKL096W CWP1 X X X Cell wall mannoprotein thank Mehul Ganatra for his technical assistance.
GPI anchor sites were predicted with Big-PI Predictor [7,8] using the metazoan different media (glucose, galactose, glycerol). Proteins
KLLA0B07370g YKL164C PIR1 X X X X O-glycosylated protein required for cell wall stability
learning set. Third, proteins were excluded if they contained a mitochondrial in each of the three samples were separated by 2D LC KLLA0B07392g YKL164C PIR2 X X X X O-glycosylated protein required for cell wall stability
localization sequence. This was predicted using TargetP 1.1 [9] with the default and identified by mass spectrometry as described [1]. KLLA0B07447g YJL158C CIS3 X X Mannose-containing glycoprotein constituent of the cell wall
non-plant settings.