SUPPLEMENTAL DATA

Enzymatic basis for N-glycan sialylation: structure of ST6GAL1 reveals conserved and unique features for glycan sialylation

L. Meng, F. Forouhar, D. Thieker, Z. Gao, A. Ramiah, H. Moniz, J. Seetharaman, S.

Milaninia, M. Su, R. Bridger, L. Veillon, P. Azadi, G. Kornhaber, L. Wells, G. Montelione, R. Woods, L. Tong, and K.W. Moremen

Supplemental Figure 1. Sequence alignment of mammalian sialyltransferases and dendrogram derived from the sequence alignment

Supplemental Figure 2. Sequence of the codon optimized ST6GAL1 fusion protein

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FLVLFIFLTSFFLNYSHTMVA

VWFLSVAFLLVFIMSLLFTYS

CNLLLAL LFLVLGFLYYSAW

CLLLKDILK TLLVFGVWILYIL

LLAMLALVLVVMVWYSIS

CLVAIFLSAVFLYYVLH ILWG

CFFWLLLLLTAA SGLLFALYF

VASVLGLVMLSVALLILSLISYVSL

SWMLAALTLLVVFLIFA

C CGASAL VVVL WLYIFPVY

ILYSSNSASAVFVILFALITIL

CWTI TISLLLIFY

CLLFIFI AFALVTLLQQILYG

CGGALLALLASLLLLLLLRLLW

C CLAV LALTTM TSLLLVYSSL

C CCLVLIIL SVVFSAVYILL W

SVIAVSFIAAFLFLLVVRLV

VQWSLLLAVLVFFLFALPSFI

MLFGIFAWGLLFLLIFIYFT

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ST6GAL1 1 ...............................................................................................................MIHTNLKKK KEKKKGSYYDSFKLQTKEFQVLKSLGKL

ST6GAL2 1 ................................MKPHLKQWRQR DSNPAEPVPSSLSFLETRRLLPVQGKQRAIMGAAHEPSPPGGLDARQALPRAHPAGSFHAGPGDLQKWAQSQDGFEHKEFFSSQVGRKSQSAFYPEDDDYFF

ST6GALNAC3 1 .....................................................................................................................................................................

ST6GALNAC4 1 .....................................................................................................................................................................

ST6GALNAC5 1 .....................................................................................................................................................................

ST6GALNAC6 1 .....................................................................................................................................................................

ST8SIA1 1 .....................................................................................................................................................................

ST8SIA6 1 .....................................................................................................................................................................

ST8SIA5 1 .....................................................................................................................................................................

ST8SIA4 1 .....................................................................................................................................................................

ST8SIA2 1 .....................................................................................................................................................................

ST8SIA3 1 .....................................................................................................................................................................

ST6GALNAC1 1 MRS LWR RHLSQG KEPQTKPSRHQRTENIKERSLQSLAKPKSQAPTRARRTTIYAEPVPENNALNTQTQPKAHTTGDRGKEANQAPPEEQDKVPHTAQRAAWKSPEKEKTMVNTLSPRGQDAGMASGRTEAQSWKSQDTKTTQ

ST6GALNAC2 1 .....................................................................................................................................................................

ST3GAL6 1 .....................................................................................................................................................................

ST3GAL4 1 .....................................................................................................................................................................

ST3GAL5 1 ...........................................................................................................................MRTKAAG AERRPLQPRTEAAAAPAGRAMPSEYTYVKLRSD

ST3GAL3 1 .....................................................................................................................................................................

ST3GAL2 1 .....................................................................................................................................................................

ST3GAL1 1 .....................................................................................................................................................................

ST6GAL1 55 AMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVVWNKDSSSKNLIPRLQKIWKNYLSMNKYKV.............SYKGPGPGIK...........................FSAEALR H.LRDHVNVSMVEVT.....................

ST6GAL2 134 AAGQPGWHSHTQGTLGFPSPGEPGPREGAFPAAQVQRRRVKKRHRRQRRSHVLEEGDDGDRLYSSMSRAFLYRLWKGNVSSKMLNPRLQKAMKD...........................YLTANKHGVR RGKREAGLSRAQLL QLRSRARVRTLDG....T

ST6GALNAC3 1 ....................MA ILKRK N........................................................................EVNFPLLLN GQPGTKWIPFSYT....................

ST6GALNAC4 1 ..................MKAPGR AGLP........................................................................LCLAT LDHH PTGSRPTVPGPLH....................

ST6GALNAC5 1 ................MKTLMRHG GGQKERPPQQQQQQQQQQQ.........................................................QASATGSSQPAAESSTQQRPGVPAG...................

ST6GALNAC6 1 ...............MA SRPPSQ EPTSLPPGPPAGRRHLPLSRRRREMSSNKEQR ................................................. NEV HYGSLRGRSRRPVN...................

ST8SIA1 1 .......MSPCGRARRQTSRGAMAVLAWKFPRTRLPM RLPNEKEIVQGVLQQG...............................................TAWRRNQTAARA RKQMED DPAHLFAMTKMN.......SPMGKS

ST8SIA6 1 .......MRP PADAPGRARILVEESREATHGTPAALRTLRSPATAVPRATNSTYLNEKSLQLTEK KNLQYGIESFSNKTKGYSENDYLQIITDIQS PWKRQAEEYAN RAKLAS DAVQNFVVSQNN.......TPVGTN

ST8SIA5 1 ..MRYADPSANRDLLGSRT RNYIKRYFEFYEGPFEYNSTR LELRHEILEVKVLSMVKQSELFDRWKSLQM K...........................WAMNISEANQ KSTLSR NAPAFLFTTQKN.......TPLGTK

ST8SIA4 1 .............MRSIRKR KTKEIARTEEHQETQLIGDGELSLSRSLVNSSDKIIRKAG...........SSIFQHNVEG...........................WKINSSLVLEIRKNILRFLDAERDVSVVKS.......SFKPGDV

ST8SIA2 1 .............MQLQFR DISEIEEEIGNSGGRGTIRSAVNSLHSKSNRAEVVINGSSSPAVVDRSNESIKHNIQPAS.......................SKWRHNQTLSLRIRKQILKFLDAEKDISVLKG.......TLKPGDI

ST8SIA3 1 ........MRN KMAR KKENIFTTPKYASPGAPRMYMFHAGFRSQFALKFLDPSFVPITNSLTQELQEKPS........................KWKFNRTAFLHQRQEILQHVDVIKNFSLTKN......SVRIGQLM

ST6GALNAC1 166 GNGGQTRKLTASRTVSEKHQGKAATTAKTLIPKSQHRMLAPTGAVSTRTRQKGVTTAVIPPKEKKPQATPPPAPFQSPTTQRNQRLKAANFKSEPRWDFEEKYSFEIGGLQTT PDSVKIKASKSLWLQKL LPNLTLFLDSRHFNQSEWDRLEHFAPPFGFMEL

ST6GALNAC2 1 ...........MGLPRGS SAVQRYPGPAAGARDTTSFEAFFQSKASNSWTG...........KGQA RHLLHLA..........................IQRHPHFRGL NLSIPVLLWGDLFTPALWDRLSQHKAPYGWRGL

ST3GAL6 1 ......................MRGY TNVYWVAPVEMKRRNKIQP LSKP...............AFASLLRF...........................HQFHPFL AADFRKIASLYGSDKFDLP................Y

ST3GAL4 1 ................MVSKSRWK REDRYIELFYFPIPEKKEP LQGEAESKA.............SKLFGNYSRD...........................QPIFLRLEDY WVKTPSAYELPYG....................

ST3GAL5 43 SRPSLQWYTRAQSKMRRPS KLNYTTEE DMKKMHYVDPDHVKRAQKYAQ..........QVLQKE RPKFAK...........................TSMALLFEHRYSVDLLPFVQKAPKDSEAES........KYDPPF

ST3GAL3 1 ...........MGLLVFVR KLHLLQWEEDSNSVVLSFDSAGQTLGSEYDRLGFLLNLDSKLPAELATKYANFSE...........................GA KPGYASALMTAIFPRFSKPAPMFLDDSFRKWARIREFVPPF

ST3GAL2 1 ................MK SLR HHSMATLPYLDSGALDGTHRVKLVPGYAGLQRLSKERLSGKS A RR MGD...........................AGASDWFDSH DGNISPVWTRENMDLPPDVQRWWMMLQPQFKSH

ST3GAL1 1 ..............MVTLRKRTLKVLT TTWFPKQMVLELSENLKRLIKHR.............P T TH IGQ...........................RKLSAWFDER NQTMQPLLTAQNALLEDDTYRWWLRLQREKKPN

ST6GAL1 158 DFPFNTSEWEGYLPKESIRTKAGP....... R . LMNSQLVTT.EKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRK....................LHPNQP

ST6GAL2 268 EAPFSALGWRRLVPAVPLSQ HPRG...... R . IINSQILTNPSHHFIDSSLYKDVILVAWDPAPYSANLNLWYKKPDYNLFTPYIQHRQ....................RNPNQP

ST6GALNAC3 54 .YRRPLRTHYGYINVKTQEP QLD....... R . .VVSHTSVPLLLKNPDYFFKEANTTIYVI G.............PFRNMRKDGNGIVYN...........MLKKTVGIYPNAQ

ST6GALNAC4 56 ........FSGYSSVPDGKP VREP...... R . .VVSHTSVPLLLRNYSHYFQKARDTLYMV G.............QGRHMDRVLGGRTYR...........TLLQLTRMYPGLQ

ST6GALNAC5 74 .....PRPLDGYLGVADHKP KMH....... R . .VIAHSSIQRILRNRHDLLNVSQGTVFIF G.............PSSYMRRDGKGQVYN...........NLHLLSQVLPRLK

ST6GALNAC6 83 ..LKKWSITDGYVPILGNKT PSR....... R . .VVAHSSVFRVLRRPQEFVNRTPETVFIF G.............PPSKMQKPQGSLVR............VIQRAGLVFPNME

ST8SIA1 105 MWYDGEFLYSFTIDNSTYSLFPQATP.FQLP R VTANPSIIRQR..FQNLLWSRKTFVDNMKI N............HSYIYMPAFSMKTGTEP..........SLRVYYTLSDVGA

ST8SIA6 152 MSYEVESKKEIPIKKNIFHMFPVSQPFVDYP R VTINPSIITLKYG..NLKEKKALFLEDIAT G............DAFFLLPAFSFRANTG..........TSFKVYYTLEESKA

ST8SIA5 130 LKYEVDTSGIYHINQEIFRMFPKDMPYYRSQ R VTVNPSIITER..FHKLEKWRRPFYRVLQV EN............ASVLLPAFYNTRNTD..........VSIRVKYVLDDFES

ST8SIA4 108 IHYVLDRRRTLNISHDLHSL PEVSPMKNRR R . ITMNPSVVQRAFGGFRNESDREKFVHRLSMLN............DSVLWIPAFMVKGGEK...........HVEWVNALILKNK

ST8SIA2 123 IHYIFDRDSTMNVSQNLYEL PRTSPLKNKH R . VTMNPSVIQRAFEDLVNATWREKLLQRLHSLNG............SILWIPAFMARGGKE...........RVEWVNELILKHH

ST8SIA3 128 HYDYSSHKYVFSISNNFRSL PDVSPIMNKH R . TTFNPSILEKYYNNLLTIQDRNNFFLSLKKLDG............AILWIPAFFFHTSAT.........VTRTLVDFFVEHRGQ

ST6GALNAC1 331 NYSLVQKVVTRFPPVPQQQL LASLPAGSLR R . YGFTAFSLTQSLLILGNRGFKNVPLGKDVR LHFLEG......TRDYEWLEALLMNQTVMSKNLFWFRHRPQEAFREALHMDRY

ST6GALNAC2 118 SHQVIASTLSLLNGSESAKLFAPPRDTP.PK R . YGFTVNTMKNSLVSYWNLGFTSVPQGQDLQ IFIPS......DIRDYVMLRSAILGVPVP..EGLDKGDRPHAYFGPEASASKF

ST3GAL6 86 GMRTSAEYFRLALSKLQS D FDEFDN..IP R . .LFYPESVFS....DPIHNDPNTTVILTA KP...........HDLRWLLELLMGDKIN.....TNGFWKKPALNLIYKPYQI

ST3GAL4 90 .TKGSEDLLLRVLAITSSSIPKNIQS...LR R . .LFYPESAH...FDPKVENNPDTLLVLVA K...........AMDFHWIETILSDKK......RVRKGFWKQPPLIWDVNPKQ

ST3GAL5 163 GFRKFSSKVQTLLELLPEHD PEHLKA..KT R . .MTYPEGAP....LSDLEYYSNDLFVAVL KS...........VDFNWLQAMVKKETLP..FWVRLFFWKQVAEKIPLQPKHF

ST3GAL3 128 GIKGQDNLIKAILSVTKEYR TPALDS..LR R . .ITYPEGAMQ....RPEQYERDSLFVLAG K...........WQDFKWLKYIVYKERV....SASDGFWKSVATRVPKEPPEI

ST3GAL2 123 NTNEVLEKLFQIVPGENPYRFRDP.....HQ R . H.FMYPESA........KNLPANVSFVLVP ..............KVLDLLWIASALST....GQIRFTYAPVKSFLRVDKEKV

ST3GAL1 112 NLNDTIKELFRVVPGNVDPM.LEKRS...VG R . H.LVYPESF........RELGDNVSMILVP ..............KTIDLEWVVSAITT....GTISHTYIPVPAKIRVKQDKI

ST6GAL1 294 FYILKPQMPWELWD LQEISPEE....IQPNP LP........SKRKTDV Q....KFFDSA TMG....AYH KNLVKH NQGTDEDIYLLGKATLPGFRTIH ..........

ST6GAL2 406 FYILHPKFIWQLWD IQENTKEK....IQPNP IP........SVRQTEL E....LYYDAA TLG....AYH KLLVQR NMGTQGDLHRKGKVVLPGFQAVH PAPSPVIPHS

ST6GALNAC3 185 IYVTTEKRMSY DG FKKETGKD..RVQSGSY INDTY .KTEGYRKVPY QGR.DE DEYFLHEHAPY.GGH KKVFAKWAKKHRIIFTHPNWTLS..................

ST6GALNAC4 181 VYTFTERMMAY DQ FQDETGKN..RRQSGSF VSDSY .REKSHPSVPY KGRLDE QMYLAHEQAPR.SAH KAVFSRWAKKRPIVFAHPSWRTE..................

ST6GALNAC5 201 AFMITRHKMLQFDE FKQETGKD..RKISNTW VPPDF R.DPNHPSVPY PFGPDE TMYLSHERGRKGSHH KRVFKNWARTFNIHFFQPDWKPESLAINHPENKPVF.....

ST6GALNAC6 212 AYAVSPGRMRQFDD FRGETGKD..REKSHSW VPPNY SQRPRLQRMPY PKGPDE VTYIQNEHSRKGNHH KRVFSSWAQLYGITFSHPSWT....................

ST8SIA1 245 NQTVLFANPNFLRS GKFWKSRG....IHAKR WPFSVN...MHEQPISH N........VLPFSG.....FH FLQLWY HKIGALRMQLDP EDTSLQPTS............

ST8SIA6 293 RQKVLFFHPKYLKD ALFWRTKG....VTAYR WPFS...KTVEDIPVSH N........KLPKHG.....FH YSQILQ HMKGILKLQFSK EVA..................

ST8SIA5 271 PQAVYYFHPQYLVN SRYWLSLG....VRAKR WAFPMN...PSGLYITH N........VKPRPG.....FH IFNFLH HSRGILRVHTGT S ..................

ST8SIA4 249 LKVRTAYPSLRLIHAVRGYWLTN...KVPIKR WPFP...KDLNGKAVKY D....LKYRYFSNASP.....H FKTLNV HNRGALKLTTGK VKQ..................

ST8SIA2 264 VNVRTAYPSLRLLHAVRGYWLTN...KVHIKR WPFPLD...QNQNPVKY S....LKYGYTSQASP.....H FKALKS HEQGALKLTVGQ DGAT.................

ST8SIA3 271 LKVQLAWPGNIMQH NRYWKNKH....LSPKR WPFG..FDPNTREDLPY K.....KGTKFTTKWQES...H FQLLYRMHGEGLTKLTLSH A....................

ST6GALNAC1 489 LLLHPDFLRYMKNRFLRSKTLDG..AHWRIYR IT.......EGHERFSD T....SWKRLIFYIN......H REVWKR HDEGIIRLYQRPGPGTAKAKN.............

ST6GALNAC2 273 KLLHPDFISYLTERFLKSKLINT..HFGDLYM ITS.......NYWKFSD R....KMKPLIFYAN......H AALWRD HKAGILQLYQR.......................

ST3GAL6 227 RILDPFIIRTAAYE LHFPKVFP..KNQKPKH KYN......FSDLKSPL N....ATMSLMNKN.....AYH QLFLKDIIEKNLVINLTQD......................

ST3GAL4 229 IRILNPFFMEIAADKLLSLPMQQ..PRKIKQK GYPD.....AYNKKQTI Q....ITLKSMAGSG......H ALAIKRMLEMGAIKNLTSF......................

ST3GAL5 307 RILNPVIIKETAFD LQYSEPQSRFWGRDKNV GYD......LNQPRTPL S....Q MAAMNFQT.....MH TKFLLK VKEGVVKDLSGGIDREF.................

ST3GAL3 270 RILNPYFIQEAAFT IGLPFNNG..LMGRGNI GYD......MSTPNAPL T....VRMAAIKESWT.....H KEFLRK VKARVITDLSSGI.....................

ST3GAL2 255 QIYNPAF.FKYIHDRWTEH.........HGRY GA........DSRGNWH .......NNRYAGEFRKT.GVH AHIIDM AKASKIEVYRGN......................

ST3GAL1 245 LIYHPAF.IKYVFDNWLQ.........GHGRY GA........DSKGNWH .......NNPSAGAFRKT.GVH SNVTAT ASINKIRIFKGR......................

Sialylmotif L

Sialylmotif S VSIII

Supplemental Fig. 1a

Supplemental Fig. 1b

Supplemental Figure 1. Sequence alignment of mammalian sialyltransferases and dendrogram derived from the sequence alignment: The full length sequences of the twenty human GT29 sialyltransferases were aligned using Geneious software (version 6.1.5, Biomatters, Auckland, New Zealand) using the MUSCLE alignment algorithm (panel A). Sequences are highlighted in color if >50% of the sequences were conserved or if the sequences were within the four sialylmotifs (gray highlights for nonconserved residues within the sialylmotifs). Transmembrane segments, as identified by UniProt, are highlighted with white text on black background. The stem regions between the transmembrane segment and the catalytic domains can not be well-defined across the family because of lack of conservation in sequence length or sequence similarity. All Cys residues are highlighted with black text on a yellow background. Residues in ST6GAL1 that were found to interact with the Galβ1,4GlcNAc acceptor in the MD simulation studies are indicated with a blue diamond above the sequence. Residues in ST3GAL1 that interact with its Galβ1,3GalNac acceptor based on the crystal structure (25) are indicated with the blue diamond below the sequence. The His base is indicated with the orange diamond in both sequences. The catalytic domain sequences were extracted from the human sialyltransferases as well as rat ST6GAL1, pig ST3GAL1, and Campylobacter jejuni CstII and aligned as above. An unrooted tree was generated using the Jukes-Cantor distance model and the Neighbor-Joining method in the Geneious software (panel B). The dendrogram shows the distinct clades of the ST6GAL, ST8SIA, ST3GALNAC (two clades) and ST6GAL (two clades) subfamilies of enzymes and the distant sequence similarity to the bacterial GT42 CstII enzyme.

ATGAGACTGCTGACAGCCCTGTTCGCCTACTTCATCGTGGCCCTGATCCTGGCCTTCAGCGTGTCCGCCAAGAGCATGCACCACCACCATCACCATCATCACatgtccggcctaaacgacatcttcgaag

M R L L T A L F A Y F I V A L I L A F S V S A K S M H H H H H H H H M S G L N D I F E

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

130

cccagaaaatcgaatggcatgaaATGagcaaaggcgaagaactgtttaccggcgtggtgccgattctggttgaactggatggcgatgttaatggccataagtttagcgtgcgcggcgaaggcgaaggcga

A Q K I E W H E M S K G E E L F T G V V P I L V E L D G D V N G H K F S V R G E G E G D

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

260

tgcgaccaacggcaaactgaccctgaagtttatttgcaccaccggcaaactgccggtgccgtggccgaccctggtgaccaccctgacctatggcgtgcagtgctttagccgctatccggatcacatgaaa

A T N G K L T L K F I C T T G K L P V P W P T L V T T L T Y G V Q C F S R Y P D H M K

88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130

390

cgccatgatttctttaagtccgcgatgccggaaggctatgttcaggaacgcaccattagctttaaagatgatggcacctataagacccgcgcggaagtgaaatttgaaggcgataccctggtgaaccgca

R H D F F K S A M P E G Y V Q E R T I S F K D D G T Y K T R A E V K F E G D T L V N R

131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173

520

ttgaactgaaaggcattgacttcaaagaagatggcaacattctgggccataaactggagtacaatttcaacagccataacgtgtatattaccgcggacaaacagaagaacggcatcaaagcgaatttcaa

I E L K G I D F K E D G N I L G H K L E Y N F N S H N V Y I T A D K Q K N G I K A N F K

174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217

650

aatccgccataatgtggaagatggcagcgtgcagctggcggatcactatcagcagaataccccgatcggcgatggcccggtgctgctgccggacaatcactacctgagcacccagtccgtgctgagcaaa

I R H N V E D G S V Q L A D H Y Q Q N T P I G D G P V L L P D N H Y L S T Q S V L S K

218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260

780

gatccgaatgagaaacgcgatcacatggttctgctggagtttgtgaccgcggcgggtatcactcatggcgaattcAGCAGCGAGAACCTGTACTTTCAGGGCAAGGACAGCACCTACAGCAAGCTGAACC

D P N E K R D H M V L L E F V T A A G I T H G E F S S E N L Y F Q G K D S T Y S K L N

261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303

910

CCCGGCTGCTGAAGATCTGGCGGAACTACCTGAACATGAACAAGTACAAGGTGTCCTACAAGGGCCCTGGCCCTGGCGTGAAGTTCAGCGTGGAGGCCCTGAGATGCCACCTGAGGGACCACGTGAACGT

P R L L K I W R N Y L N M N K Y K V S Y K G P G P G V K F S V E A L R C H L R D H V N V

304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347

1040

GTCCATGATCGAGGCCACCGACTTCCCTTTCAACACCACCGAGTGGGAGGGCTACCTGCCCAAAGAGAACTTCCGGACCAAAGTGGGCCCTTGGCAGAGATGCGCCGTGGTGTCTAGCGCCGGCAGCCTG

S M I E A T D F P F N T T E W E G Y L P K E N F R T K V G P W Q R C A V V S S A G S L

348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390

1170

AAGAACAGCCAGCTGGGCCGGGAGATCGACAACCACGACGCCGTGCTGAGATTCAATGGCGCCCCTACCGACAACTTCCAGCAGGACGTGGGCAGCAAGACCACCATCCGGCTGATGAACTCCCAGCTGG

K N S Q L G R E I D N H D A V L R F N G A P T D N F Q Q D V G S K T T I R L M N S Q L

391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433

1300

TCACCACCGAGAAGCGGTTTCTGAAGGACAGCCTGTACACCGAGGGCATCCTGATCGTGTGGGACCCCAGCGTGTACCACGCCGACATCCCCAAGTGGTATCAGAAGCCCGACTACAACTTCTTCGAGAC

V T T E K R F L K D S L Y T E G I L I V W D P S V Y H A D I P K W Y Q K P D Y N F F E T

434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477

1430

ATACAAGAGCTACCGGCGGCTGAACCCCAGCCAGCCCTTCTACATCCTGAAGCCCCAGATGCCCTGGGAGCTGTGGGACATCATCCAGGAAATCAGCGCCGACCTGATCCAGCCCAACCCCCCTAGCAGC

Y K S Y R R L N P S Q P F Y I L K P Q M P W E L W D I I Q E I S A D L I Q P N P P S S

478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520

1560

GGCATGCTGGGCATCATCATCATGATGACCCTGTGCGACCAGGTGGACATCTACGAGTTTCTGCCCAGCAAGAGAAAGACCGACGTGTGCTACTACCACCAGAAGTTCTTCGACAGCGCCTGCACCATGG

G M L G I I I M M T L C D Q V D I Y E F L P S K R K T D V C Y Y H Q K F F D S A C T M

521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563

1690

GCGCCTACCACCCCCTGCTGTTCGAGAAGAACATGGTCAAGCACCTGAACGAGGGCACCGACGAGGACATCTACCTGTTCGGCAAGGCCACCCTGAGCGGCTTCCGGAACATCCGGTGCTGA

G A Y H P L L F E K N M V K H L N E G T D E D I Y L F G K A T L S G F R N I R C .

564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

1812

Signal sequence His Tag

TEV site

AviTag

AviTag

“superfolder” GFP

“superfolder” GFP

“superfolder” GFP

“superfolder” GFP

superfolder” GFP

“superfolder” GFP rat ST6GAL1

rat ST6GAL1

rat ST6GAL1

rat ST6GAL1

rat ST6GAL1

rat ST6GAL1

rat ST6GAL1

rat ST6GAL1

Supplemental Figure 2. Sequence of the codon optimized ST6GAL1 fusion protein: The expression cassette encoding the ST6GAL1 fusion protein was

generated by gene synthesis and was codon optimized for expression in human cells by Geneart AG (Regensburg, Germany). The coding region sequence was

comprised of a 25 amino acid signal sequence (yellow box), 8xHis tag (blue box), AviTag (grey box), “superfolder” GFP (green box), TEV protease cleavage

site (orange box), and residues 95-403 of rat ST6GAL1 (red box) as indicated (see Experimental Procedures).