( Agrocybe aegerita)

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 19 (1) : ( Agrocybe aegerita) 3,,, ( ), AAVP( Agrocybe aegerita antiviral protein) SDS2PAGE 1518 kd 32 kd IEF2PAGE 318 AAVP N N N QGVNIYNIVAGA PDGPWLVEK AAVP 12 3, 18 3 AAVP AAVP AAVP Ca 2 + Mg 2 + Zn 2 +,AAVP SGC27901, MGC2803, BGC823 HL260 AAVP mgπkg,, Q291,Q786 Purification and Characterization of a Novel Lectin AAVP from Fruiting Bodies of the Edible Fungus, Agrocybe aegerita SUN Hui, ZHAO Chen2guang, TONG Xin, QI Yi2peng 3 ( Laboratory of Molecular Virology, College of Life Sciences, Wuhan University, Wuhan , China) Abstract A novel lectin Agrocybe aegerita antiviral protein (AAVP) was purified, from the fruiting bodies of edible fungus Agrocybe aegerita by precipitation of 40 % 80 % (NH 4 ) 2 SO 4 followed by DEAE2SepharoseFast Flow and Sephacryl S2200 chromatography The relative molecular mass of AAVP was 32 kd tested by gel fil2 tration, the subunit of AAVP was 1518 kd determined by SDS2PAGE Its p I was about 318 by isoelectric fo2 cusing AAVP is probably unglycosylated and blocked by pyroglutamyl at the N2terminus The amino acid se2 quence of the N2terminus was QGVNIYNIVAGA, and the sequence of a segment of AAVP obtained after being digested by trypsin was PDGPWLVEKR AAVP agglutinated 3 types of human and 12 types of animal erythro2 cytes regardless of blood group or category However, its hemagglutinating activity for mammalian erythrocytes is much higher than that for bird and aquatic In a hemagglutination assay, porcine mucin was the only inhibi2 tor of the lectin among the 18 sugars and 3 glycoconjugates tested AAVP was thermal2stable, acid2stable and alkali2stable Its activity was unaffected by demetalization and addition of divalent metals including CaCl 2, MgCl 2 or ZnCl 2 AAVP had inhibiting effect on tumor cell lines SGC27901, MGC 8023, BGC 823 and leuco2 : : (No ) 3 Tel : ,E2mail : edu cn, Received :June 4,2002 ; Accepted :September 9,2002 Supported by the National Natural Science Foundation of China, No Corresponding author Tel : ; E2mail : edu cn

2 1 : ( Agrocybe aegerita) 97 cythemia cell line HL260 in vitro AAVP is toxic to mouse by celiac injection with an LD 50 of mg Πkg body weight Key words Agrocybe aegerita antiviral protein, purification, characterization, hemagglutinating activity, an2 titumor activity, HL Pemberton [1 ] (1994), g, ml,50 % 100 ml 2 10 h, % 80 %, [2 ] 0101 molπl (ph 610) ( 2 ) DEAE2Sepharose FF (116 cm 10 cm) Sephacryl2200 (116 cm 80 cm) ( ) SDS2PAGE Laemmli [3 ] [12 ], 15 %, [2 ], 5 % R2250 ( ) Superdex 75HR HT29 Coca22 c ( M r 1214 kd), A ( M r 25 kd) [4 MCF27 ] ; ( M r 45 kd) IL21 IL22 TNF2 [5, 6 ] [7 ; ] [8, 9 ] [10 ] ( Catsimpoolas [13 ] ) ; HeLa ( 2 %Ampholine PAGE [11 ) ] ( 1 % Ampholine, 10 % ), 0101 molπl H 3 PO molπl NaOH h ph ph, pi DEAE2Sepharose Fast Flow Sephacryl S Pharmacia ; Promega 6 molπl HCl 110 ; Sigma ;Ampholine 18 h : D D2 D mgπl D D2 N2 20 min A 490 A 490 2D2,3 A 490, Sigma (2 %Ampholine,20 % ),200 V 10 h R2250 ; (1 cm) 1 ml 10 mmolπl KCl, ( ) N : ; N2 N2 SGC27901 MGC2803 BGC823 N2 N 3

3 98 19 N Applied MTT Biosystems 470A Πml 96 ( ) : HPLC gπml 100 gπml 24 h,,pbs [ ] l 115 mgπml MTT,37 V 25 l, 4 h MTT 200 l DMSO (90 %DM2 1 mgπml,25 l 2 % SO, 10 % 011 molπl 2NaOH, ph 1010),37 30 min A h, V,, :, 2 n ( n % = ( A - A )ΠA 100 % ) [ ] [ ] 20 g ; 012 molπl ( 715 mgπ, ml), ( nπ2 10, 3 d,, n ) 015 h, mgπml AAVP, 2 HCl,NaOH ph, min ph 6 DEAE2Sepha2 rose FF (0101 molπl,ph mgπml AAVP, 610),0 013 molπl NaCl min, D1,D2 ( Fig 1A) D2, Sephacryl S2200, S AAVP (Fig 1B),, 20 mmolπl EDTA 212 ; 10 mmolπl SDS2PAGE(Fig 2) IEF(Fig 3) Ca 2 + Mg 2 + Zn 2 +, 1 AAVP SDS2PAGE Fig 1 Purification of the AAVP (A) Ion2 exchange chromatography of the protein crude extract on a DEAE2Sepharose FF column (116 cm 10 cm), molπl NaCl, 1 mmolπl PB buffer (ph 615) for gradient elution (B) Gel filtration of fraction D1 on Sephacryl S2200 column (116 cm 80 cm) eluted with 25 mmolπl PB buffer

1 : ( Agrocybe aegerita) 99 1518 kd,, AAVP 32 kd AAVP 2 N2, IEF,, 10 mmolπl KCl 24 h ph, QGVNIYNIVAGA AAVP AAVP N Protein (pi) 318 N N N2 Table 1 Amino acid composition of AAVP Amino acid Content

4 1 : ( Agrocybe aegerita) kd,, AAVP 32 kd AAVP 2 N2, IEF,, 10 mmolπl KCl 24 h ph, QGVNIYNIVAGA AAVP AAVP N Protein (pi) 318 N N N2 Table 1 Amino acid composition of AAVP Amino acid Content Residues in one molecular Asx Thr Ser Glx Pro Gly Fig 2 SDS2PAGE of AAVP 1 Protein molecular mass marker ; 2 AAVP Ala Cys 0 0 Val Ile Leu Tyr Phe Met Lys His Try ND ND Arg Glx, sum of Glu and Gln ; Asx, Asp and Asn ; ND, not determined Fig 3 Isoelectric focusing of AAVP on 5 % PAGE containing Ampholine HPLC, 213 : PDGPWLVEK AAVP Table ( Trp ), Table 2 AAVP 3 ( Gln) (Asn) ( Glu) 12 (Asp) 15 Table 1,AAVP Cys (7 ) (2 ) His Met (6, ) 3,AAVP ( ) ,7 mgπml AAVP AAVP : AAVP , 214 N :AAVP SDS2PAGE

5 Table 2 Erythrocyte agglutination activity of AAVP Erythrocyte Titer of agglutination Erythrocyte Titer of agglutination Human(A type) 2 8 duck 2 5 Human(B type) 2 9 white eel 2 4 Human(C type) 2 8 turtle 2 4 Rabbit 2 10 yellow eel 2 8 Bovine 2 14 frog 2 5 Sheet 2 10 fish 2 8 Mouse 2 8 loach 2 5 Chicken 2 3 Π Π The initial concentration is 1 mgπml 2 3 = 125 gπml ; 2 4 = 6215 gπml ; 2 5 = 3113 gπml ; 2 8 = 3191 gπml ; 2 9 = 1195 gπml ; 2 10 = 0197 gπml ; 2 14 = 0106 gπml The same as the below table Table 4 ph 16,AAVP ( D2 D2 (ph 2142) (ph 12122) D2 1 D D2 N2 2D2 ) Table 4 Stability of AAVP under different acid or basic conditions (100 mmolπl) AAVP ; 3 (715 mgπml) AAVP 7132 gπml,aavp mmolπml EDTA AAVP ; 10 ; , Table 3 Thermal stability of AAVP Temperature Titer of agglutination activity Room trmperature The initial concentration is 1 mgπml c (acid or base)πmol L - 1 ph value Titer of agglutination NaOH, NaOH, NaOH, NaOH, HCl, HCl, HCl, HCl, The initial concentration is 1 mgπml mmolπl Ca 2 + Mg 2 + Zn 2 +,AAVP 2110,AAVP Table 5,AAVP 4 3 HL Ara2C Table 3 AAVP ;, 100,AAVP gπml SGC ,AAVP Table 5 The inhibition effect of AAVP on tumor cells ( %) SGC27901 MGC 8023 BGC 823 HL gπml 50 gπml Inhibition of AAVP at different concentration 10 gπml 5 gπml 1 gπml Ara2C 100 gπml P < 0101,compared with cells treated by Ara2C

6 1 : ( Agrocybe aegerita) 101 Ara2C ; MGC8023 BGC823 Ara2C Table 6 Toxic activity of AAVP to mouse 10 gπml 3 50 % Fig 4,AAVP (1 10 gπml), > 10 gπml AAVP AAVP AAVP AAVP Ticha (1985) [16 ] AAL 44 kd 22 kd, Fig 4 The inhibition ratio of AAVP of different concentration on tumor cells 2111 Mo (2000) [17 ] Polyporus squamosus,aavp PSL ( Polyporus squamosus lectin),n [15 ] PFEGHGIYHIPSVNTANVRI AAVP Table 6 r = 1 N ( Fig 5), 33, lgld 50 = 1 2, lg = mgπkg 20 g,ld mgπkg 3 DoseΠ lg[ dose ] mg kg - 1 Total number Dead number 50 ( ), AAVP AAVP min (ph ), of mice of mice Dead ratio ( %) Probability Π unit Π 2 AAVP AAL,AAVP,AAVP 3 AAVP AAVP,AAVP, AAVP AAVP AAVP AAVP N protein AAVP Western AAVP Fig 5 Comparison of the N2terminal sequences of AAVP and PSL

7 AAVP N [7 ] 1217 kd,,,n Met His Cys ; [18 ] 26 kd,n [8, 9 ] 1214 kd,n, 2 % AAVP [18, 19 ] LD mgπkg AAVP LD mgπkg AAVP 3 (SGC27901 MGC 8023 BGC 823), AAVP [20 ] AAVP 3 AAVP N AAVP AAVP cdna, AAVP People s Press), 1986 ; AAVP ( References) 1 Pemberton R T Agglutinins (lectins) from some British higher fungi Mycol Res, 1994, 98 : Wang H X, Bun T N, Ooi V E C Lectins from mushrooms Mycol Res, 1998, 102 (8) : Peumans W J,Van Damme J E M Lectins as plant defense protein Plant physiol, 1995,109 : Yu L G, Fernig D J, Smith J A, Milton J D, Rhodes J M Reversible inhibition of proliferation of epithelial cell lines by Agaricus bisporus (edible mushroom) lectin Cancer Res, 1993, 53 : Licastro F, Morini M C, Kretz O, Dirheimer G, Creppy E E, Stripe F Mitogenic activity and immunological properties of bolesatine, a lectin isolated from the mushroom Boletus satanas Lenz Int J Biochem, 1993, 25 : Ennamany R, Kretz O, Badoc A, Deffieux G, Creppy E E Effect of bolesatine, a glycoprotein from Boletus satanas, on rat thymus in vivo Toxicology, 1994, 89 : Ko L, Hsu C I, Lin T H, Kao C L, Lin J Y A new fungal immuno2 modulatory protein, FIP2fve isolated from the edible mushroom, Flam2 mulina velutipes and its complete amino acid sequence Eur J Biochem, 1995, 228 : Haak F M, Kino K, Sone T, Jardieu P Ling Zhi28 : A novel T cell mi2 togen induces cytoking production and upregulation of ICAM21 expres2 sion Cell Immunol, 1993, 150 : Van2Der2Ham L G, Van2Der2Vliet J A, Bocken C F M, Kino K, Hoitsma A J, Tax W J M Studies on a new immunomodulating agent Transplantation (Baltimore), 1995, 60 : Wang H X, Ng T B, Ooi V E C, Liu W K, Chang S T Actions of le2 tins from the mushroom Tricholoma mongolicum on macrophages, splenocytes and life2span in sarcoma2bearing mice Anticancer Res, 1997, 18 : Kawagishi H, Nomura A, Mizuno T, Kimura A, Chiba S Isolation and characterization of a lectin from Grifola frondosa fruiting bodies chim Biophys Acta 1990, 1034 : Bio2 12 Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature, 1970, 227 : Catismpoolas N Microisoelectric focusing in polyacrylamide gel col2 umns Anal Biochem, 1968, 26 : , : ( Sun Ce, Zhu Zheng, Mo Han2qing ed Lectins Beijing : Science Press), : ( Xia Bing2 nan ed Handbook of Pharmaceutical Experiment Guiyang : Guizhou 16 Ticha M, Dudova V, Kocoruek J Studies on lectins L A nonspe2 cific erythroagglutinating lectin and a blood2group A specific lectin in the mushroom Agrocybe aegerita (Brig ) Sing In : Bog2Hansen T C and Breborowicz J ed Lectins2Biology Biochemistry, Clinical Biochemisty, Berlin :Walter de Gruyter, 1985 : Mo H Q, Winter H C, Goldstein I J Purification and characterization of a Neu5Ac 226Gal 124GlcΠGlcNAc2specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus J Biol Chem, 2000, 275 (14) : Hsu H C, Hsu C I, Lin R H, Kao C L, Lin J Y FIP2vvo, a new fun2 gal immunomodulatory protein isolated from Volvariella volvacea chem J, 1997, 323 : Bio2 19 Lin J Y, Chou T B Isolation and characterization of a lectin from edible mushroom, Volvariella volvacea J Biochem, 1984, 96 : : ( Ying J Z eds Illustrated Handbook of Chinese Medicinal Fungi Beijng : Science Press), 1987

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