Quantitative proteomics reveal the anti-tumour mechanism of the carbohydrate recognition domain of Galectin-3 in Hepatocellular carcinoma Mingchao Wang, Fang Tian, Wantao Ying*, Xiaohong Qian* Correspondence to: QIAN Xiao hong, Email:
[email protected] Wan tao, Email:
[email protected] Affiliations: Beijing Institute of Radiation Medicine, National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing, China Tel/Fax: +86-10-80705055; +86-10-80705155 Running title: Anti-tumor mechanism revealed by quantitative proteomics Key words: Hepatocellular carcinoma, Quantitative proteomics, Gal3C
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A >Avi-Gal3C ATGGCTGGGCCACTGATTGTGCCTTATAACCTGCCTTTG CCTGGGGGAGTGGTGCCTCGCATGCTGATAACAATTCTG GGCACGGTGAAGCCCAATGCAAACAGAATTGCTTTAGA TTTCCAAAGAGGGAATGATGTTGCCTTCCACTTTAACCC ACGCTTCAATGAGAACAACAGGAGAGTCATTGTTTGCAA TACAAAGCTGGATAATAACTGGGGAAGGGAAGAAAGAC AGTCGGTTTTCCCATTTGAAAGTGGGAAACCATTCAAAA TACAAGTACTGGTTGAACCTGACCACTTCAAGGTTGCAG TGAATGATGCTCACTTGTTGCAGTACAATCATCGGGTTA AAAAACTCAATGAAATCAGCAAACTGGGAATTTCTGGT GACATAGACCTCACCAGTGCTTCATATACCATGATAGGA CTGAATGACATCTTCGAAGCACAGAAAATCGAGTGGCA TGAG
B >Sequencing result of Avi-Gal3C ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACT GGTGGACAGCAAATGGGTCGCGGATCCGAATTCATGGCTGGGCCACTGATTGTGCCTTATAACCTGCCTTTGCCTG GGGGAGTGGTGCCTCGCATGCTGATAACAATTCTGGGCACGGTGAAGCCCAATGCAAACAGAATTGCTTTAGATT TCCAAAGAGGGAATGATGTTGCCTTCCACTTTAACCCACGCTTCAATGAGAACAACAGGAGAGTCATTGTTTGCAA TACAAAGCTGGATAATAACTGGGGAAGGGAAGAAAGACAGTCGGTTTTCCCATTTGAAAGTGGGAAACCATTCAA AATACAAGTACTGGTTGAACCTGACCACTTCAAGGTTGCAGTGAATGATGCTCACTTGTTGCAGTACAATCATCGG GTTAAAAAACTCAATGAAATCAGCAAACTGGGAATTTCTGGTGACATAGACCTCACCAGTGCTTCATATACCATGA TAGGACTGAATGACATCTTCGAAGCACAGAAAATCGAGTGGCATGAGCTCGAGCACCACCACCACCACCACTGA
C >rGal3C MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMAGPLIVPYNLPLPGGVVPRMLITILGTVKPNANRIALDFQR GNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKL NEISKLGISGDIDLTSASYTMIGLNDIFEAQKIEWHELEHHHHHH
Supplementary Figure S1. Informtion of pET28a-AG and rGal3C.(A) Plasmid pET28a-AG was synthesized by inserting the 468 bp Avi-Gal3C DNA fragment into plasmid pET28a. (B) Nucleotide sequencing results of the inserted DNA fragment(Avi-Gal3C), the DNA fragment encoding carbohydrate recognize domain of Galectin-3 was underlined. (C) Amino acid sequence of rGal3C. The first 36 amine acids were the N-terminal histione tag (HisHisHisHisHisHis) and T7 tag(MetAlaSerMetThrGlyGlyGlnGlnMetGly), and underlined amine acids from
37-177 were the core sequence of carbohydrate recognition domain of galectin-3, and amine acids from 178-200 were the avi-tag(GlyLeuAsnAspIlePheGluAlaGlnLysIleClu) and C-terminal histione tag (HisHisHisHisHisHis). 2
IPAMTIAK
LLSELDQQSTEMPR
13 13 Supplementary Figure S2. Incorporation of C6-L-lysine and C6-L-arginine into HepG2 at various time points. The peptide IPAMTIAK (Mr=843.49) has a charge state of 2 and contains one lysine, the peptide LLSELDQQSTEMPR (Mr=1645.84) has a charge state of 2 and contains one arginine, and their heavy isotope is 6.032 Da higher than the light isotope. The ratio of intensity of heavy to light peak steadily increased. On day 21, this peptide was fully incorporated with 13 C6-L-lysine or 13C6-L-arginine.
3
A
Photo-Leucine labeled rGal3C
B UV fixation
With UV fixation and galactose
Without UV fixation
C
241
341 147
64
D
Overlap proteins 164
30
Transmembrane proteins
Number of transmembrane domains MOT4 12 GTR1 S38A1 10 S12A3
4F2
8
LAT1
AT1B1 CALX CD166 ITA1 ITA2
6 4
HACD3
ITAV
2
ITA3
ITB1
0
EGFR
L1CAM
BASI
LAMP2
ADT3
LCAP
TFR1 STOM PTPRJ
LRP1
PTN
NPTN
MPRD MPRI MRC2
Supplementary Figure S3. (A) The Photo-Leucine labeled rGal3C was detected by 12% SDS-PAGE. (B) The binding of Photo-Leucine labeled rGal3C to HepG2 cell surface with UV fixation, without UV fixation or with UV fixation in the presence of lactose. (C) Overall 241 and 341 proteins were identified in two independent repeated experiments respectively, and 177 were repeated identified. In the 177 overlap proteins, 30 transmembrane proteins analyzed by TMHMM 4
Server v2.0 were considered as potential receptors of rGal3C. (D) A radar map displayed the number of transmembrane domians of the 30 potential receptors of rGal3C. Supplementary table S1 List of up-regulated proteins Protein IDs P09455 Q9UJX2 P31949 Q9NX74 O95470 Q6PUV4 Q14764 P37802 O75746 Q9BQL6 P09382 P15559 Q56P03 P01011 P52943 O43301 Q92887 Q9Y3A2 P38571 P31947 P53634 Q14914 O14908 Q8N1I0 P56747 Q9UGH3 P10644 O60443 Q99519 O75312 Q8IYU8 Q00653 P06280 P36955 P48506 O60678 Q13509 P13284
Gene names RBP1 CDC23 S100A11 DUS2 SGPL1 CPLX2 MVP TAGLN2 SLC25A12 FERMT1 LGALS1 NQO1 EAPP SERPINA3 CRIP2 HSPA12A ABCC2 UTP11L LIPA SFN CTSC PTGR1 GIPC1 DOCK4 CLDN6 SLC23A2 PRKAR1A DFNA5 NEU1 ZPR1 MICU2 NFKB2 GLA SERPINF1 GCLC PRMT3 TUBB3 IFI30
unique peptide number
SILAC ratio
H/L
L/H
H/L
L/H
1 3 2 2 9 4 8 3 3 2 4 8 1 6 3 2 10 3 3 8 7 4 2 3 1 1 9 6 4 5 5 3 2 5 10 2 4 2
2 1 2 1 10 2 5 2 2 3 4 9 2 6 2 2 9 2 6 7 6 4 2 2 3 1 8 5 3 3 3 3 3 7 12 3 4 2
5.46 1.31 5.16 1.08 1.13 2.93 2.54 1.78 1.03 1.72 2.87 1.29 1.01 3.21 1.47 1.78 1.01 1.01 1.11 1.45 1.12 1.07 1.07 1.04 2.08 1.23 1.13 1.10 1.91 1.06 1.19 1.19 1.03 1.19 1.14 1.21 2.01 1.78
3.23 3.05 2.43 2.30 2.05 2.05 2.03 1.97 1.87 1.86 1.85 1.84 1.81 1.81 1.79 1.77 1.76 1.76 1.74 1.72 1.68 1.68 1.66 1.65 1.63 1.63 1.61 1.59 1.54 1.54 1.53 1.53 1.53 1.52 1.51 1.51 1.46 1.44
5
P00966 P60981 P01009 O60218 Q16658 P23368 P04792 Q16643 Q13501 P21333 Q01995 Q9NR12 Q99536 Q9NRZ9 Q14807 Q8IYB7 P01034 Q16881 P67936 Q9HC07 Q7Z406 P51948 P52292 Q5TFE4 P78504 P46821 Q9UK76 P42575 P35579 P02794 Q9H0A8 Q01664 Q9UPY5 Q9ULT8 P04035 O14530 Q9Y371 P31153 P06400 P46013 Q99439
ASS1 DSTN SERPINA1 AKR1B10 FSCN1 ME2 HSPB1 DBN1 SQSTM1 FLNA TAGLN PDLIM7 VAT1 HELLS KIF22 DIS3L2 CST3 TXNRD1 TPM4 TMEM165 MYH14 MNAT1 KPNA2 NT5DC1 JAG1 MAP1B HN1 CASP2 MYH9 FTH1 COMMD4 TFAP4 SLC7A11 HECTD1 HMGCR TXNDC9 SH3GLB1 MAT2A RB1 MKI67 CNN2
10 7 17 16 12 3 5 6 6 66 9 6 10 5 4 3 3 12 5 2 3 1 6 8 3 3 2 2 44 3 6 1 1 11 6 4 6 5 5 17 7
11 9 17 16 12 2 2 6 2 67 6 4 12 8 5 4 2 14 4 2 2 1 6 8 2 3 1 1 33 2 2 2 2 3 2 4 2 2 3 15 6
6
1.71 1.50 1.54 2.31 2.02 1.56 2.56 1.72 2.59 1.54 5.26 1.73 1.57 1.53 1.56 1.57 1.55 1.79 1.50 1.68 1.83 1.67 1.85 1.69 1.68 1.93 1.77 1.53 1.91 1.91 1.52 1.68 2.47 1.63 2.50 1.50 1.70 1.60 1.58 1.63 1.74
1.39 1.38 1.35 1.33 1.31 1.29 1.23 1.20 1.18 1.16 1.15 1.15 1.15 1.15 1.14 1.14 1.13 1.13 1.12 1.11 1.10 1.10 1.10 1.10 1.09 1.09 1.08 1.07 1.06 1.05 1.05 1.04 1.04 1.03 1.03 1.02 1.01 1.01 1.01 1.00 1.00
Supplementary Table S2 List of down-regulated proteins Protein IDs Q08AH3 Q92597 P10909 P48029 P37059 P08833 P05121 P0C7P0 P23141 P08319 P02671 Q06033 P35575 P21549 O00584 Q16877 Q13740 Q02338 P02787 Q6UWM9 Q4G0N4 Q8N6C5 P55268 P13674 P07148 Q9Y646 P52569 P56181 P10646 Q9C0E8 Q8TEQ8 P51687 P09972 Q9BQE5 Q93088 Q9UDR5 Q2QL34 Q02252 Q969G6 Q9UN36 Q96I45
Gene names ACSM2A NDRG1 CLU SLC6A8 HSD17B2 IGFBP1 SERPINE1 CISD3 CES1 ADH4 FGA ITIH3 G6PC AGXT RNASET2 PFKFB4 ALCAM BDH1 TF UGT2A3 NADK2 IGSF1 LAMB2 P4HA1 FABP1 CPQ SLC7A2;SLC7A1 NDUFV3 TFPI LNP PIGO SUOX ALDOC APOL2 BHMT AASS MPV17L ALDH6A1 RFK NDRG2 TMEM141
unique peptide number
SILAC ratio
H/L
L/H
H/L
L/H
2 9 11 3 5 3 9 1 26 11 9 6 1 9 7 5 6 8 30 8 6 14 6 15 9 4 2 1 6 7 2 2 9 4 6 15 2 16 2 7 2
3 9 6 3 6 4 12 2 27 9 8 2 1 9 9 4 7 7 28 6 5 8 4 17 8 3 1 1 6 4 1 1 10 5 5 8 2 16 1 5 1
0.55 0.37 0.29 0.71 0.86 0.58 0.59 0.61 0.95 0.62 0.42 0.32 0.30 0.84 0.46 0.57 0.74 0.50 0.61 0.67 0.60 0.46 0.70 0.52 0.61 0.63 0.67 0.43 0.58 0.60 0.50 0.74 0.64 0.82 0.48 0.70 0.73 0.62 0.70 0.63 0.64
0.22 0.38 0.53 0.25 0.21 0.32 0.34 0.33 0.27 0.43 0.64 0.85 0.94 0.35 0.64 0.54 0.41 0.63 0.53 0.48 0.54 0.72 0.49 0.66 0.58 0.57 0.55 0.88 0.65 0.65 0.77 0.54 0.62 0.49 0.84 0.58 0.56 0.66 0.59 0.66 0.66
7
Q9UBM7 P16591 Q96EY8 Q96A26 Q15120 Q8NBP7 Q96HE7 P08754 P00450 P28332 Q16134 Q9BY49 P00734 P16662 P78549 Q86WA6 Q16850 Q00577 Q99424 Q9BPW8 O00468 P39060 O95479 Q16762 Q15118 P05204 Q6JQN1 O14638 Q04446 Q8N4Z0 O00560 P00387 Q9UKR5 O00469 Q96QD8 P02679 Q969Z3 O95573 P46019 O75600 P31327 Q7Z3D6 Q13011 Q9NR19
DHCR7 FER MMAB FAM162A PDK3 PCSK9 ERO1L GNAI3 CP ADH6 ETFDH PECR F2 UGT2B7 NTHL1 BPHL CYP51A1 PURA ACOX2 NIPSNAP1 AGRN COL18A1 H6PD TST PDK1 HMGN2;HMGN3 ACAD10 ENPP3 GBE1 RAB42 SDCBP CYB5R3 C14orf1 PLOD2 SLC38A2 FGG MARC2 ACSL3 PHKA2 GCAT CPS1 C14orf159 ECH1 ACSS2
3 5 4 6 1 3 12 2 8 9 8 11 9 4 1 8 15 5 6 7 16 6 7 4 2 2 2 3 9 2 1 8 2 16 6 13 3 17 6 5 21 2 10 13
2 2 3 4 1 4 13 3 5 10 6 12 4 5 1 6 12 4 5 9 11 6 4 5 1 2 2 5 11 1 5 7 3 18 5 14 4 18 7 3 10 1 8 11 8
0.47 0.81 0.61 0.66 0.60 0.74 0.58 0.46 0.75 0.65 0.76 0.55 0.49 0.95 0.52 0.54 0.84 0.71 0.67 0.65 0.63 0.74 0.63 0.63 0.96 0.86 0.55 0.59 0.65 0.78 0.53 0.62 0.62 0.60 0.50 0.63 0.80 0.89 0.66 0.80 0.61 0.59 0.65 0.81
0.90 0.53 0.71 0.65 0.73 0.59 0.76 0.97 0.60 0.69 0.59 0.83 0.93 0.48 0.88 0.86 0.55 0.66 0.71 0.73 0.75 0.65 0.77 0.77 0.51 0.57 0.88 0.84 0.77 0.64 0.94 0.81 0.81 0.85 1.00 0.80 0.63 0.57 0.77 0.64 0.84 0.87 0.79 0.65
P05026 Q9Y5U8 Q5T2W1 P04080 P02771 Q13825 Q9H0W9 P50453 Q9NPJ3 Q15345 Q53GQ0 P07305 Q9H6V9 P61225 P35914 Q8N4T8 P53365 Q96BR5 Q8TE04 Q008S8 O00264 Q96AB3 Q9P0M6 Q9H7C9
ATP1B1 MPC1 PDZK1;PDZK1P1 CSTB AFP AUH C11orf54 SERPINB9 ACOT13 LRRC41 HSD17B12 H1F0 C2orf43 RAP2B HMGCL CBR4 ARFIP2 COA7 PANK1 ECT2L PGRMC1 ISOC2 H2AFY2 AAMDC
9 1 9 6 17 3 6 11 3 2 12 7 3 2 6 4 2 4 1 1 4 3 13 3
8 1 8 5 22 5 9 7 4 4 14 6 5 1 9 3 2 2 3 1 3 3 12 5
9
0.66 0.61 0.57 0.89 0.67 0.63 0.58 0.92 0.64 0.66 0.59 0.63 0.65 0.65 0.62 0.59 0.92 0.66 0.93 0.94 0.66 0.66 0.63 0.67
0.81 0.87 0.94 0.60 0.80 0.85 0.93 0.58 0.85 0.83 0.93 0.89 0.87 0.87 0.92 0.97 0.62 0.89 0.63 0.64 0.94 0.96 1.00 0.97
Supplementary Table S3 List of potential receptors of rGal3C
Protein ID P08195 P26006 P56199 Q01650 P17301 P05026 P35613 P27824 Q13740 P00533 P06756 P32004 P13473 Q9UIQ6 P02786 P12236 P11166 Q9P035 P05556 Q07954 O15427 P20645 P11717 Q9UBG0 Q9Y639 P21246 Q12913 P55017 Q9H2H9 P27105
Gene Unique Unique iBAQ_1 iBAQ_2 Name peptides_1 peptides_2 4F2 13 15 51388000 102170000 ITA3 10 12 8734800 28183000 ITA1 5 12 2471600 8907500 LAT1 4 4 27689000 31059000 ITA2 3 4 868750 3969000 AT1B1 2 3 1045300 28864000 BASI 2 3 9130800 20833000 CALX 2 3 979220 2728000 CD166 2 5 816130 4586500 EGFR 2 6 1298000 5759300 ITAV 2 5 145470 2540000 L1CAM 2 6 423470 5865000 LAMP2 2 2 1830100 8407500 LCAP 2 1 398200 322830 TFR1 2 1 352510 316730 ADT3 1 1 1446800 13671000 GTR1 1 2 2255600 3853300 HACD3 1 2 1331300 7679700 ITB1 1 5 0 11314000 LRP1 1 1 53056 93367 MOT4 1 1 15661000 11441000 MPRD 1 1 1747600 2610500 MPRI 1 8 333210 1091100 MRC2 1 1 167160 888230 NPTN 1 2 430810 2348500 PTN 1 1 1882300 25552000 PTPRJ 1 5 427780 2018200 S12A3 1 1 301770 446250 S38A1 1 2 3849200 9622400 STOM 1 3 859220 6564700
10
