0 10 20 30 40 50
Retention time (mim)
Figure I‑3. PurirlCation of Spanish mackerel egg lectin by ion‑
exchange chromatography on a Hi･Trap Q column.
Elution bdfer : 0 to 1.0 M NaCI Iineargradient in 20 mM Tris･
tt tt [0 8E 盲3 3t n! q･ ZO Sq V
ut t0 97 一t
!S df tV W'
〇
Table Ill. PurirlCation of Spamish mackerel egg lectin
Purificationstep & FV問 ﾖr彦 F ﾆ 7F庸宥 &V6 fW'窒R V6貿 7F庸宥 W&貿 F柳詛 ﾆB
Extract 鉄 ﾃ3S ﾃ #Bﾃ #
L‑RhammoseSepharose4B 緜33"ﾃ 33 ﾃS鼎"ﾃ塔r
Hi‑Trapq 纉#Sbﾃ #Sモﾃ#sc"ﾃイ
a: Obtained by multiplying the hemagglutination titer against 2% rabbit erythrocytes by the totalvolume of the solution.
b: Total activity/mg of protein
‑1̲̲̲tl 免ﾆﾂ
･̲凍■ ｨﾆ"ﾒ
F i g
･ 2 F i g u r e H . S D S
･ P A G E o f S M L u n d e , n o n
･ , e d u c i n g c o n d i t i o n , SD S･ PA GE wa sc ar ri ed ou to n1 5% ge l･
m
a
r
k
e
r
S
h
t
L
Figure I･6･ Determination of the molecular mass of SML by MALDI･
TOF mass spectrometry.
Mod● A oprLtIOn
&LrdorL曲 PDl帆ty 血叩けiboJ) ￠ntml
A瓜drLtltlr YEIlL▲P Grld l巾LtAP GudJ VJreO ExtrACtIOn仙y tape
Ant)inthrL rrLLA rLnP
Ntlmbr a l■■●r ■bLJL h●肝LTltJnrlty LJ●■ Jtep hL4
Lin●■r Dtl■yld Po■ltlY●
Muu■1
26DOO V 肘ヽ 0 16I1 300 n■●亡
10000 ･1 100000 DL IOOIJPCtrun 27師 きOth CLl)brltJOTt tWe bt吋爪LI
J)仙y■pyDJLt▲Y打lmeWq+)YO30227yttrIJIYlrL.tJIJnlmyOFlobLn Cat G■LLLIrLbn rEL▲trJ3E Sllt■plluC ■qd
LdW Ttltu Pb lCdO DL
DIPLIIr ltArt LAn+
ELITt JL7Je
NtlTnbF OHJLLJL PlTttJ
V亡rLJCLI ICLh.
Vertbl out:
Input bJdwldLh
S■nFIIe vdl PILte TD StrlLl mlmbtr lnrtruTtl■nt TtLme PhtE type mrJt&rr)e tAb nJLme
Ab●dllt■ X.P■LtJt}rL Ah■dtlEe rPOJLLJOn RthtLVtは･PCLILJOrt RdLtlYC y POtlLltOll SltotJI Ln LFI●dnLm SDtl Etie prt‑tITE MLrrV T汀erJtlrt VC2 TITeLLt)rl TIS ptA･ VJJdLh TIS ALght L叩Fth
8858 4 rLAeC 17869 100 mV Oヽ 2占htth
70 STIMEISOSI I 1222
Voy LPrDE ･m
C YVOYAGERVIOOwdl pILte P】t PE B10■1lAtemB
17307 8 17716 5 0 22278 919 OOl loo I 565e･007 I 788pOO8 0 002081 15 11832
Fi gu re t･ 5･ SD S･ PA GE of SM La ft er re du ct io nw it h2
･
m e r c a p t o e t h a n o l ･ S D S ･ P A G E w a s c a r t i e d o u t o n 1 5 % g e I ･
marker SML
Table I･2･ IJIhibition of hemagglutinatib activity of SML by saccbarides･
a Minimum concentradon of saccharides required for complete
inhibition
b D‑Mannose, trehalose, D‑N‑ace(ylglucosamine･
c Glycoproleins such as fetuin and mucin type ll.
0 1 0 20 30 40 5￠
RetetLtion time (min)
Figure I‑8. GeI爪Itration chromatograplly Of SML on a Superdex1
200 column (1.6 X 300 mm) in 20 mM Tris‑HC) / 0.5 M NaCl (pH t m
O S
= t
t !
S A
f t
V S
O .
0
‑.+lY 辻ｲ 耳 霹 宥Dﾂ粐ﾂﾔﾂﾒ謦ﾒ ｣､ "ﾂﾅ ｹ ﾂ謦ﾂ&停簇撞｢ﾃ｢粢 ﾆ ﾉ y ｩyR
盛憲 ;悼,,潔 唸 7 ｨ ﾂ
0 5 10
DTBP (mg/d)
Mo no mer
Figure I･9･ SDS･PAGE of SMI, cross‑linkedwith a homo
bifunctional reageBL
SML was dissolved in 50 LL 1 of0.1 M biethanolamine (pH
8.0) at a concentradon of 0.25 mg/ml and reactedwith50 fL I
of dimethyll3,3 '‑di‑thio‑his propionimidate (DTBP) (5 ･0, 10･0 mg/mi) in 0. I M triethanolamine. The reaction wasallowedto proceed for I h at room temperature and stopped by adding
Fig. 3
0 10 20 30 40 50 60 70
Retemtiom time (mim)
Figu･e I‑Ill Separation of peptides generated by digestiotI Of CAM･
SMLwith AchTOmObacter protease I. peptides were separated by reversed‑
phase HPLC on a Wakosi1 5C4‑200 coluTTn (5 Jl m, 4･6 X 250 mm) using a gradient of
acetonibile in 0.1% TFA. Flow rate was 1 ml/min.
tu tt OZ el dS an V9 巾. I
(%) at!J)!tXO一33V
0 0 5 4
0 1 0 20 30 40 50 60 70
Retention time (min)
Figtlre I‑12･ Separation Of peptides generated by digestion of CAMI SMLwith S･ aweus V8 protease. peptides were separatd by reversed‑phase HPLC on a Wakosi1 5C4‑200 column (5 LE m, 4.6 X 250 mm) using a gradient of
0 1 0 20 30 40 50 60 70
Retemtiom time (mim)
Figure I‑13. Separation of peptides generated by digestion or
deglycosylated CAM･SMLwith endoproteinase Ar貰‑C. peptides we1℃t t m O e = I t
! S d n V 8 r T
(%) 3t!h一!uO一33V
3 0 加
tuttON=一t!SiLLtVS寸.0 (%) atPT!tXO133V
O
0
3 ' 一
o l0 20 30､ 40 50 60 70
Retention tiJne (min)
Figure I･14･ Separation of peptides generated by digestion or deglycosyhted CAM‑SMLwitb S･ aweus V8 proteikSe･ Peptides were separated by reversed‑phase HPI･C on a Wakosi1 5C4‑200 coILlmn (5 FL m, 4･6 X 250
0 10 20 30 40 50 60 70
Retemtiom time (mim)
Figure I･15･ Separation of peptides getlerated by digestion Of PE‑SML
with trypsin･ Peptides were sera.ated by reversed‑phase HPLC on a TSKge1 0DS120TcoluTrLn (5 IL m, 4.6 X 250 mm) using a gradient ofaceloni山Le in O･1% TFA･ Flow ttTtt0巾=ltFS巴‑VO6JO (%) 3tPtttTOt33V
洲 e r q
ttJtt07=lt!SdLtV寸9.0 (%) aTP一P〇一33V
O
0
3 2
0.90 AtJFS at 220 nn
'..I
L+. '..i..̲ヨ
㌔
デー,..̲萱. I
◆2
㌔ ●
㌔.
〜..≡ゴ星■'..喜tg
･G..‑A
■巴.+
窒'..
&'...
■■1■1
○ ⊆ 皆 旨 さ 皆
Acetonitrile (%)
lVH土HnCEIG…VJIAL岳cEHGVHVq℡▲LYGREt)さ)℡CtZlGKBLQQ工S‑ZiC8LLGAVDYL‑FteDGKkVCELS‑IFRJ,gZ),CSDTYKYLQTKYMT
LrH柵.'L帆}' 1 i‑1‑ n[･I.れn･Lp I A ‑=rn...‑‑ l ｣
1m".棚.M I.̲lT.1:ニ】;17.;器,‑" l I
I 1‑‑" '帆川M･O' l I I‑‑ 】"‑ a".･･ l I 7.‑ユ1ニ‑･･ ･･‑.･･ l Lここ巴H‑ Ll‑･1･
｣̲ヱヒ二二二二⊥二土.二････.･.･.JTW‑1‑:j･‑･こ.･ ‑.･l m.I; I"‑ ･..‑=∴･い l
l v‑･‑‑‑b, [
110 1M l加 140 1帥 l̀○ ▼ 1‑I) 180 190 200
HzYLV℡CZipSVAHt･HCDYGCVISVYNADYlZ…DX℡℡CBYERVPSQZQNRDCSTT,℡8KVAZi8CSQ‑WCHIJLSNLVPGD,CVGIY7(YLEVAYVCqYfISIV
l ‑ ⊥y･‑‑‑A‑つ‑ I
I ve‑lJ‑日脚ム' l vt一日…J.】州,≡, l
:lL･如･! l けL‑】?.TMl m･!き芦〉 l
L.･N･:･.‑ l一･二･L‑･ l Th･1.‑ A‑I ...r･1. 1 J m‑"〜さ" 1 Im=‑ I ITW一日l･"･‑‑三･l
Fig. 4
I lけ‑lTmH nきよHl
Figure I･17. Summary of sequence detemination of SMI..
Lys‑X: CAM‑SML digested withAchromobacter protease I; V8‑X: CAM‑SML digested withS. aureus
V8 protease; RN‑X: deglycosylated CAM‑SML digested withendoproteinase Arg‑C; VN‑X: deglycosylated
CAM‑SML digested withS･ aureus V8 protease; TRP‑X: PE‑SML digested withtrypsin : TRN‑X:deglycosylated CAM‑SML digested withtrypsln.
Values are the molecular masses measured by MALDI‑TOP mass spectrome叩･ Values in parenthesesare
fromthe sequences･ Thearrowhead indicatesthe glycosylation site.
F i g u r e t ･ ) 6 , S e p a r a t i o n o f p e p t i d e s g e n e r a t e d b y d i g c s t i o n o f
deg)ycosylatedCAM･S空戸withtrypsin,peptidesweresepaTatedbyreversed･
ph as eH PL Co na Wa ko si 15 C4
･2 00 co tu mA (5 1L m1 4. 6X 25 0m m) us in ga gr ad ie nt of
a c e t o n i b i l e i n O . 1
% T F A
̲ F t o w r a t e w a s l m u m i n .
2 0 3 0 4 0 5 0 6 0 7 0
Re te nt io nt im e( mi n)
0 10 20 30 40 50 60 70 80 90
Retentiom time (巾im)
Figure II･23･ Neutral and amiJ10 Sugar analysis of SML･
PyridylamiJIO derivadves of the hydrolysate of SML were subjected to anion‑exchange chromatography I
Fig. 5
0 10 20 30 40 50 60 70 80 90
Relemtiom time (min)
Figure IIl24･ NedraJ and amino sugaranalysis･
Pyridylamino derivatives of the standard sugar.were subjected to anion‑
exchange chromatography･ GalNAc: PAIN‑acetylgalactosamine, Xyl:
PAIXylose･ GIcNAC: PA‑N‑acetylglucosamine, Glu: PA‑glucose, Man:
3 3 t t 3 3 S a h O n t a
3 3
t t
3 3
S 3
1 0
n T
a
0 1 0 20 30
Retemtiom time (mim)
Figure II125. Sialic acid analysis of SMI..
DMB derivatives of the hydroIysate of SML were subjected
to reversed‑phase HPLC.Fig. 6
0 1 0 20 30
Retention time (min)
Figure IIl26. Sialic acid analysis.
DMB derivatives of the staJldard sialic acid were subjected to
reversed‑phase HPLC･ NGNA: DMBIN‑glycolylneuramimic 33tr 33 S3 JO nu
3 3 t t 3 3
$ 3
h O
n T
d
0 20 40 60 80
Retemtiom time (仙in)
Figtlre II‑5. Separation OE the adsorbed fraction from ConAI
Sepharose chromatography･ The digest of CAM‑SML widl
Achronwbacter protease l was subjected to ConA‑Sepharose andtheadsorbed丘ac也on was separated by reversed‑phase tIPLC om a
TSKgel ODS 120T colurrm (5 LL m, 4.6 A 250 mm) using a gradient of
Fig.7
0 20 40 60 80
Retemtion tiJne (min)
Figure IIJ. Separation of the tLnadsorbed fraction from CoIIA･
Sepharose chromatography. The digest of CAM‑SML with
AchromobacteTI PrOteaSe I was subjected to ConAISephaJ10Seandtheunadsorbed fraction was separated by reversed‑phase HPLC on a TSKge1 0DS 120T colunu (5 FL m, 4.6 X 250 mm) using a gradient of
ttTttONT盲S巴‑V=CO
(% )3 T! Jt
!t t0 )3 3V
4 0 3 0 加
t m O =
= I t ! S a L t V N e . 0
( % ) 3 ] t J I
! u O 7 3 3 V
4 0 3 0 2 0
Yoyur恥ITP)I I Qe5.9. 10暮■7)
Figure II･7･ MALDI･TOF mass spectrum of the adsorbed peptide on ConA･
Sepharose in negative･ion mode.
Fig. 8
【M+H】+
Vqtpr Spc JltBP F257Bj. 11か9】
167 187