High-yield novel leech hyaluronidase to expedite the. preparation of specific hyaluronan oligomers
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1 High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers Peng Jin 1,2,3, Zhen Kang 1,2,3,6, Na Zhang 1,2,3, Guocheng Du 1,3,5,6 & Jian Chen 1,3,4,6 1 Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi , P. R. China. 2 The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi , P. R. China. 3 School of Biotechnology, Jiangnan University, Wuxi , P. R. China. 4 National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi , P. R. China. 5 The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi , P. R. China. Synergetic Innovation Center of Modern Industrial Fermentation, Wuxi , P. R. China. Correspondence and requests for materials should be addressed to Z. K. ( zkang@jiangnan.edu.cn) or to 6 J. C. ( jchen@jiangnan.edu.cn). Phone: , Fax:
2 Supplementary Table S1 Primes used in this study Prime SMART II oligonucleotide 5-3 sequences AAGCAGTGGTAACAACGCAGAGTACGCGGG oligo(dt) (T) 18 GSDF GSDR GSPR UPM long UPM short HaseF HaseR LHyalF H4LHyalF H6LHyalF H9LHyalF H12LHyalF LHyalR GCTTTTACTATCCAYCASTATTA TTAWTTTTTGCAKGCWTCAACGTTAGCATC AGCGTCAAGGTATGTTGACACATCTGAGG CTAATACGACTCACTATAGGGCAAGCAGTGGTAACAA CGCAGAGT CTAATACGACTCACTATAGGGC ATGAAAGAGATCGCGGTGACAATTGACG TTATTTTTTGCAGGCTTCAACGTTAG CCGGAATTCATGAAAGAGATCGCGGTGACAATTGAC CCGGAATTCCACCACCACCACATGAAAGAGATCGCG GTGACAATTGAC CCGGAATTCCACCACCACCACCACCACATGAAAGAG ATCGCGGTGACAATTGAC CCGGAATTCCACCACCACCACCACCACCACCACCAC ATGAAAGAG CCGGAATTCCACCACCACCACCACCACCACCACCAC CACCACCACATGAAAGAG TCCGCGGCCGCTTATTTTTTGCAGGCTTCAACGTTAG C 2
3 Supplementary Table S2 ESI negative ion species of HA oligosaccharides HA Molecular mass [M-H] - [M-H] 2- [M-H] 3- [M-H] 4- oligomer HA HA HA HA HA HA HA
4 Supplementary Table S3 The effects of metal ions on the LHyal activity Metal ions (1 mm) Relative activity (%) Control 100 K ± 2.6 Na ± 5.8 Mg ± 2.5 Fe ± 9.9 Zn ± 6.5 Ni ± 3.4 Ca ± 5.9 Cu ± 3.1 Co ± 4.5 Mn 2+ ND a The effects of metal ions on LHyal activity. The residual enzymatic activity was measured after incubation with various metal salts at final concentrations of 1 mm under standard conditions. An enzyme reaction in buffer only was used as a measure of 100% activity. a ND not detectable. 4
5 Supplementary Figure S1 The chemical structure of hyaluronic acid and classification of hyaluronidases. The polymer consists of repeating disaccharide units that are linked by β-1,3 glycosidic bonds; the disaccharide units consists of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) linked by β-1,4 bonds. This classification of hyaluronidases was based on both substrate specificity and biochemical analyses of the hyaluronidases and their reaction products. Supplementary Figure S2 Multiple sequence alignment of LHyal with mammalian hyaluronidases. The figure was performed using the Clastal W1.8 software package based on the known hyaluronidases from Mouse HYAL-1 (NP_032343), Homo sapiens HYAL-1 (NP_149349), Rattus norvegicus HYAL (AAD01980). The conserved residues are marked with * and boxed in yellow column except for two predicted key catalytic residues that are colored red. Semi-conserved sites are boxed in pale blue column. Supplementary Figure S3 The colorimetric determination of N-acetyl-d-glucosamine at the reducing end of oligosaccharides liberated from hyaluronic acid by Morgan Elson reaction. Reaction buffer: alkaline borate solution (dissolving 1.73 g H 3 B 3 and 0.78 g K in 10 ml water, 1/10 volume of 0.8 g ml -1 K 2 C 3 added before use), p-dimethylaminobenzaldehyde solution (2 g p-dimethylaminobenzaldehyde dissolved in 2.5 ml of concentrated HCl and 7.5 ml glacial acetic acid, diluted with 4 volumes of glacial acetic acid before use). The reaction solution of LHyal and BTH 400 μl were added 110 μl of alkaline borate solution and heating 4 min in boiling water, then added 1.5 ml of p-dimethylaminobenzaldehyde solution and incubated at 37 C for 20 min, Control was set using HA solution without enzyme. Supplementary Figure S4 The mass spectrum profile of BTH hydrolysis products by LC-MS-IT-TF. The reaction mixture containing 1.6 mg HA and 2,700 U BTH in per milliliter 50 mm citrate buffer (ph 5.5) were incubated at 38 C for 1 h 5
6 (a) and 8 h (b). The anion species of HA2, HA4, HA6, HA8, HA10, HA14 (larger oligosaccharides can not be detected) were detected at the initial and latter of the reaction stage by BTH. Supplementary Figure S5 The HPSEC MALLS RI system was used to determine molecular weight of HA oligomers with reaction 1 h (Fig. 4a) by LHyal. SEC and LLS chromatograms of HA oligomers in 0.2 M aqueous NaCl at 30 C detected by laser light scattering photometry (LS: red line) and differential refractometer (RDI: blue line). According to the curve analysis, the Mw (weight-average molecular weight), Mn (number-average molecular weight) and Mp (peak position molecular weight) values were , , and , respectively, and Mw/Mn = Such a result indicated that the distribution of HA oligomers molecular weight were a main relatively concentrated peak by size-exclusion chromatography. HPSEC MALLS RI system consists of a pump (515 HPLC, Waters, USA), an Rheodyne 7725i injector (Waters, USA) with a 200 μl injection loop (Rheodyne, IDEX Corporation, USA), two HPSEC columns, a MALLS detector (Wyatt Technology DAWN HELES, USA), and a RI detector (ptilab Rex, Wyatt Technology Co., USA). Supplementary Figure S6 Identification of LHyal protein molecular weight with WATERS MALDI SYNAPT Q-TF MS (QTof Ultima, Waters). (a) The total ion chromatogram (TIC) of the water-soluble LHyal protien isolated by LC analytical column (BEH C mm 1.7 μm). (b) The mass chrommatogram of multi-charged cationic species corresponding to the part of LHyal protien TIC (8.48 min, red line in a). (c) Single-chargd mass chrommatogram of LHyal protien calculated according to multi-charged m/z in (b). The database analysis gave a single significant peak with the molecular mass of 58,684.0 Da for LHyal. Supplementary Figure S7 MS 3 mass spectra with corresponding structures of [M-H] -. The precursor ion [M-H] - (red arrow labeled) is bombarded by 6
7 argon to generate two fragment ions in MS2 mass spectra. The two fragment ions [M-H] - (green arrow labeled) and [M-H] - (orange arrow labeled) as the precursor ions are further bombarded to generate [M-H] - and [M-H] - in MS3 mass spectra, respectively. The formulas of fragment ions are calculated and corresponding structures are predicted with the LCMS postrun software. 7
8 C CH 2 C CH 2 C CH 2 C CH 2 NHCCH 3 GlcNac GlcUA NHCCH 3 n EC Bovine testicular hyaluronidase EC Leech hyaluronidase CH 2 C NHCCH 3 NHCCH 3 C CH 2 C CH 2 C NHCCH 3 CH 2 NHCCH 3 repeating disaccharide EC Bacterial hyaluronate lyase NHCCH 3 NHCCH 3
9 . * : :.. :... Mouse MLGLTQHAQKVWRMKPFSPEVSPGSSPATAGHLLRISTLFLTL-LELAQVCR GSVVS N 57 Homo MAGH L LPICALFLTL-LDMAQGFR GPLLP N 29 Rattus MR AA GLGPIIT L ALVLEVAWASELK PTA PPIFT G 33 LHyal ---MKEIAVTIDDKNVIASVSESFHGVAFD SLFSPKGLWSFVDITSPKLF K LLEGLSPGYFRVGGTFA N * :.. : : : : * :. * Mouse RPF ITVWNGD---THWCLTEYGVDVDVSVFDVVANKEQSFQGSNMTIFYREELG--TYPYYTPTGEPVF G 122 Homo RPF TTVWNAN---TQWCLERHGVDVDVSVFDVVANPGQTFRGPDMTIFYSSQL GG --TYPYYTPTGEPVF G 94 Rattus RPF VVAWNVP---TQECAPRHKVPLDLRAFDVEATPNEGFFNQNITTFYYDRL --LYPRYDAAGMSVH G 98 LHyal WLF FDLDENNKWKDYWAFKDKTPETATITRRWLFRKQNNLKKETFDDLVKLTKGSKMRLLFDLNAEVRT G Mouse Homo Rattus LHyal Mouse Homo Rattus LHyal Mouse Homo Rattus LHyal Mouse Homo Rattus LHyal Mouse Homo Rattus LHyal Mouse Homo Rattus LHyal **** : :. : :... :.:. :.: GLPQNASLVTHLAHTFQDIKAAMPEPDFSGLAVI DWEAWR-PRWAFNWDSKDIYRQRSMELVQAEHPDW P 191 GLPQNASLIAHLARTFQDILAAIPAPDFSGLAVI DWEAWR-PRWAFNWDTKDIYRQRSRALVQAQHPDW P 163 GVPQNGSLCAHLPMLKEAVERYIQTQEPAGLAVI DWEEWR- PP VWVRNWQEKDVYRQSSRQLVASRHPDW P 167 YEIGKKMTSTWDSSEAEKLFKYCVSKGYG--DNI D WELGNE DHTSAHNLTEKQVGEDFKALHKVLEKY P :.. ::..... : : ETLVEAAAKNQFQEAAEAWMAGTLQLGQVLRPRGLWGYYGFPDCYNNDFLS--LNYTGQCPVFVRDQNDQ 259 APQVEAVAQDQFQGAARAWMAGTLQLGRALRPRGLWGFYGFPDCYNYDFLS--PNYTGQCPSGIRAQNDQ 231 SDRIVKQAQYEFEFAARQFMLNTLRYVKAVRPQHLWGFYLFPDCYNHDYVQNWDSYTGRCPDVEVAQNDQ 237 TLNKGSLVGPDVGWMGVSYVKGLADGAGDHVTAFTLHQYYFDGNTSDVSTYLDAT Y FKKLQQLFDKVKDV * * :.. :..:. :.. : : ::. : : LGWLWNQSYALYPSIYLPAALMGTEKS--Q MYVRHRVQEALRVAIVSRDPH-VPVMPYVQIF-YEMTDYL 325 LGWLWGQSRA L YPSIYMPAVLE GT GKS--QMYVQHRVAEAFRVAVAAGDPN-LPVLPY VQIF-YDTTNHF 297 L AWLWAENTALFPSVYLDKTLASSKHS-- RR NFVSFRVQEALRVAHTHHANHALPVYVFTRPT-YTRRLTE 304 LKNSPHKDKPLWLGETSSGYNSGTKDVSD YVSGFLTLDKLGLSAANNVKVVIRQTIYNGYYGLLDKNTL :..: * :.. :.* * * :.... LPLEELEHSLGESAAQGVAGAVLWLSSDKTSTKESCQAIKAYMDST LGPFIVN--VTSAALLCSEALCSG 393 LPLDELEHSLGESAAQGAAGVVLWVSWENTRTKESCQAIK EYMDT T LGP F ILN--VTSG ALLCSQALCSG 365 LNQMDLISTIGESAAL G SAGVIFWGDSVYASSMENCQNL KK KYLTQTLVPYIVN--VSWATQYCSWTQCHG 372 EPNPDYWLMHVHNSLVGNTVFKVDVSDPTNKARVYAQCT TNSKH T QSRYYKGSLTIFALNVGDEDVTLK : :. :....*.. * :. : : : : HGRCVRHPSYPEALLTLNPASFṠIELTHDGR PP SLK--GTLSL-KDRAQMAMKFRCRCYRGWRG---KWC 457 HGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLR--GALS L -EDQAQMAVEFKCRCYPGWQL---TVD 429 HGRCVRRNPSASTFLHLSPSSFRLVPGRTPSEPQLRPEGELSE-DDLS YLQMHFRCHCYLGWGGEQCQWN 441 IDQYSGKKIYSYILTPEGGQLTSQKVLLNGKELKL VSDQLPELNA D ESKTSFTLSPKTFGFFVVSDANVE DKRGM VSVDQV HKRAAGDASRAWAGAHLASLLGLVAMTLTWTL 473 ACKK * * * * * * *
10
11 a Inten.(x100,000) HA HA HA HA HA HA10 HA m/z b Inten.(x1,000,000) 6.0 HA HA HA8 HA HA HA HA HA m/z
12 LS DRI
13 a 0.97 TF MS ES+ TIC 2.69e5 % b 100 Time Time (8.84) TF MS ES % m/z c 100 Time (8.84) TF MS ES % mass
14 MS1 (E-) Inten. (x10,000,000) CH 2 4 C 3 CH 2 2 C e- 1 -H HNCCH 3 HNCCH m/z MS2 (E-) Precursor: Inten. (x1,000,000) e CH 2 CH 2 C 3 CH 2 -H HNCCH 3 HNCCH 4 3 HNCCH m/z C e- -H MS3 (E-) Precursor: Inten. (x100,000) e- C 3 -H m/z MS3 (E-) Precursor: Inten.(x100,000) C CH HNCCH m/z e- -H
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