N型CTIEASNLVFGDPCVGIYK

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

15 30 45 60 75 90

RetentioJl time (min)

Fi卯re II･13･ HPLC chromatogram of PA･sugar chains derived froJn

SML otm TSKgeI ODS 120T column (4.6 I 250 mm).

Fig. 9

0 1 5 30 45 60 75 90

Retemtion time (min)

Figure II･14･ HPLC chromatogram of PA･glucose oligomers (DP =

3‑22) om a TSKgel ODS 120T coJumn (4.6 Ⅹ 250 mm).

33 tt 33 S3 JO nu

L n 4 0

( % ) T O u q n 甲 T

33 tZ 33 S3 JO nt 4

0 1 5 30 45 60 75 90

Retemlion time (min)

Figu'e II･15･ HPLC chromatogram of PAISugar Chain 009 0n a

TSEgel OJ)S 120T column (4.6 Ⅹ 250 mm).

0 1 0 20 30 40 50 60

Retemtiom time (mim)

Figure II‑16. HPLC chromatograms of sugar chains derived from SML orL a TSKgeI Amidel80 column (4.6 X 250 mm).

33 tt 33 S3 JO TL L4

( % ) t o u q q Z ･ 1

3 2 爪

33 tT 33 S3 JO nL 4

(% )3 T! 17

!t ZO 13 3V

S 4 0

0 1 0 20 30 40 50 60

ReteJItioh time (mill)

Figure II‑17･ HPLC chJ･OmatOgram Of PA‑gltICOSe OIigomers (DP =

3‑22) ob a TSKgel Amide･80 column (4.6 Ⅹ 250 mm).

0 1 0 20 30 40 50 60

Retemtiom time (mim)

Figure II‑18. HPLC chJ.OmatOgram Of PA･sugar chain 009 0n a

TSKgeI Amide･80 ColumJl (4.6 I 250 mm).

33 t[ 33 S3 JO tt L4

(% )3 t! 11

!t t0 73 3V

′ 0 4

33 u3 3$ 3] [O nT a

(% )3 T!

･Z TP O7 33 V

爪

0 丘

V 4

Amide‑silica (glucose units)

U JL LJ, 〇､ ‑1 80 0

Rd∝tor Del■yed PdtltIVe MwLLA1

20㈱ V 67%

112 002%

125 n批

900 ‑ 1800 DL bOIq)ectruJtI 2806 30III DCELult

2.8 DlhydTOXybenLOl⊂ LCld 5仙DA

OIr hZdJl Of o叩･■t)On

Extr■don rTde PBL▲rLty AcqttIl)boTL Cmttd

Acdq･L叫yolhp qrtd vqlttp NJrrl)T VOlttp rdJO OtJlde vlrd 0 E･ELrLCtADh ddty LIErle

Ac叩由tlm JttJJT rmP NLlrrlber d lJJeE rhob hJeT JnLtttJdLI LAJtr REP RLLe CdIbTtuOrl一ype CdLbrLtI叩rrLItELゝ LAVJ mbJ PLt!

I)ned LOn比lectJ)r

DLPtlter BLJLrt hm1 44 014 BLrL暮lZA 1 ll●即 NuztlbtMrdLh pelTItJ ' 1278b Vqt4CAl他山e 0 a00mV VertlCJLI cErMt: 0ヽ

boo MTl1

88 SEIh(EJ80Sl1 4222 ヽbyLPr DE 孤 C:VVOYACER¥100 vJell J'l■tb Plt PE EhoJヽ114rrl■

37687 7 17Bl1 3 390 19 986 825 50 I 788e OO7 3 723p･008 0 001769 15 ユ1832 JTLPut b▲ndvldLh 0

S■Enpie wdI Pl■Er lD BeTILl nurrtber lrtdrlrrN rtL llArtlP PtLtb ty7n Litulttt)I LIB nlm甘

Ab8bluLe x pJltlOrl Abdtlte V･FNWltlOn Rd&tJVe 3. PdfItLqn Rd山Ve YPfltlbTL SlVLJHTI BJNdFtrm S卯TCe prE8紬re M廿rOr Pe仙tJZY TC2 pre帖ure TZS pte w)d山 TZS I)LFhL lt･npJl Vqq'r S叩■1 t･AevBCt32,0事.0 I )‑NFO 7tBP暮1町的. 0141)

Figure IIl20･ MALI)Ⅰ･TOF mass spectrum of PA･sugar chains derived

from SML. DHBA was used as a matrix.

L 4

± L2 l ○ ≡

●l̲ l

●

■

■ l

●ミ小L17]

●LKI

◆X32

● X a J

▲{ち▲2

●●▲S▲▲ ▲lO142

●

▲ 一

〇

▲

● き l

●占Pl̲

●▲09322

+)IOIr ▲︼○‑〇

++1]0.22

●貞P6]

●◆IAt2

●●10.l3

●2]023

◆

▲ 8 5 I

▲

︼ o 1 7

.2...23 T.I.,,.tl･.≡

. H 5 4 . 3 0 0

♪ 2 ] 0 2 2

◆

〜 5

‑ 2 W 4 q 2 1

▲ l O 1

● 5 0

‑

● 冨 2 . 3 1 0 1 2 1 1 1

甘墜 2. .J L1

･･ te 簸喜

● 套ゝ 3

● H S 3 . 2 1 6 m . . 2 D 1 A o b l

●X34●l示l

X3B

◆30.‑l1004

● IO).Z

一1PI

●ALZJ ｢︻

･‑

･

vI . .巨 2 3 1 tS

･ .T 0 ., 3･ 1 .. I .+

●ZdO3

‑

‑ , T . 幸 3

･ +

= ' 1 . 蔓

･ ] 1 3 . A .

･

tL O. 2

● 2 0 0 . l

●

● ] 0 ] 2

● O I P + 1 0 1

L Al

9 L O

︻ 2 1 3 1 4 7 5 ) 6 L T t 8 ) や 2 0 2 ] 2 2

0 D S ･ s i l i c a ( g l u c o s e u m i t s ) F i

g u r e t t

･ ) 9 ･ T w o J i m e n s i o n a l m a p o f P A t a g g e d s u g a r c b a i n s

･

Th eX ax is is gl uc os eu ni ts us it Lg OD S･ si li ca an dY ax is is gl ue os e

un it st ts in gA mi de あi Li ca .M ar ks lI ar et he po si

︻i on so fP A･ su ga r ch ai ns de ri ve df ro mS A4 ). .

Vq'P Spq ntJ･Adv8印も〇･5.m)t胴.TTBP LIMA. 1佃】

Figure II･21･ MALDIITOF mass spectrum oE PA･sugar chains derived from SML. DHBA was used as a matrix.

No･ structures of sugar chains

N曲doprLtl071 Eb血JnOdJ PoLJLnty Acqthltl叫でOntZd

血Ⅰ肝■hnr vdl■p Orld ydt▲p

Jhr voLtLP rLLn

Oulde ■lr■ O:

htr峨○爪ddq･ bmc

A叩)れtIOn m1‑ T仰 Nt)rrbbq oE LL8et Jhotr LdL" Lhhlty

ZAHq RIZP R▲b Cdlbr■dorL typ●

CLLlbrdlOh rnJLtrLX LOV ErIJJJ PtC 一mEELed Ion Jd肋r

DIFIbtq仙t tJme Blnれtモ NL.rRtNFr OT血Lt TnlrLh VerM ∝tIeO VertJCd o払tL;

Input bLJldvIIdtJI 0

SJlTIpr●一山1 PJtLe ZE) SerLd number lTt■ttLl巾mI nLErLE Pl■te type lLL8TtLme

hb nlme

AbjblLLte yl伽tlOn AIIWltJt. rPeLltAqTI RektJ叫Ⅹ･JWllJOn IbIAtJVe ∫ PCのtlLon 釣IBt* Ln ■脚rllm 与our伽PrCJ4ttrt MlrrOT Prt‑uTe m prEJ8ure llS pte vqdd) TIS爪ldlt len幹h

9001 18Cl)DA bqIPqtEun 2880

Lo,.":,.

2.5･Dlhy血中tyben山LC JLCld

…D■

Orr

ll 041 I lllk 12788 800 mV Oヽ aoO NTl)

68 8BIltEJSOhq1 1 4222

Vo.vAitlr.DE m

C yVOYA(;ERYIOO ､州pILte PIE PS BIVヽ1tJ rTI I

87720 2 1 7052 572 738 224 185 80 1 8丘7e･007 2 776e･008 0001828

1さ 1183 2

GIucose unit

ODS Amide‑SO Ml･

001

100.1

210.4

Manα1.6

Man β1 ･4GIcNAcβ1 ･4GIcNAe 7.2 4.3 990.20 Manα1.3

GIcNAcβ1 ･2ManoLl l6

Manβ1･4GIcNAcβ1･4GIcNAc 9.5 4.7 1 193.56 Manα1･3

Galβ1･4GIcNAcβ1･2Manα1･6 FUG α1.6

GaLPl｡G.cNAcPL2Manα1.TanP1‑4GIc"AcβL4GIc"Ac 13･8 7･4 1883･72

Figure II122･ Structures of sugar chains corresponding to the number of 2･D map･

No･00l and No･100･1are structures which are proposed for the sugarchains from SML ; No･210･4 is PA‑009 (Takara).

ど

蜘叫

仙帆

. W

m 温

伽

0 1 5 30 45 60 75 90

Retentiom time (mim)

Figtlre I1‑27･ HPLC of PAISugarChains derived from SML on a

TSKgd ODS 120T coILLmn (4.6 Ⅹ 250 mm).

The numbers onthepeaksare glucose units.

1 5 30 45 60 75 90

Retemtiom time (mim)

Figure I1‑28. HPLC of PA･glucose oligomers (DP = 3‑22) on a

TSKge1 0DS 120T column (4.6 X 250 Jnm).

The numbers onthe peaks are glucose units.

3 3 t Z 3 3 S a

･ 1 0 n T a

( % ) t o t r q q Z

･ 1

3 2 0 0

3 3 u 3 3 S 3 J O n t A

( % ) T O t Z q n t [ ･ T

j 2 0 0

0 1 0 20 30 40 50 60 70

Retemtiom time (mim)

Figure II‑29･ HPLC ofsugarchains derived from SML ob a TSKgel

AmideJO columb (4.6 Ⅹ 250 mm).

The numt"rs on the peaksare glucose units.

0 1 0 20 30 40 50 60 70

Retemtiom time (mim)

Figure IIl30･ HPLC of PA‑ducose oligomers (DP ≡ 3‑ll) on a TSKgd Amide･80 CoILLmn (4.6 Ⅹ 250 mJn).

33 t[ 33 S; 3J Ot LL 4

(% )3 IP t! uO 一3 3V 伽 4

33 tr 33 Sa

･1 0T LL 4

(% )3 t!

︼一 Pt Ot 33 V 榊 4 0 劫

Voyqer Spec刑lBP = 1723.5, 9183】

GdPIJGlcNAc β 1‑2 Mddtd･6

MAJtβl JG]cNAcβl JIGIcNAc GAIBIJGlcNAc a l･2 M姐al･3

Mr, 172132

17665687

HJt■ (17t/I)

Figure II･31･ MALDI･TOF mass spectrum of PAISugar Ctlains

derived from SML. DHBA was used as a matrix.

No･ Structures of sugar chiTtS

叫一一

器

ヨ0∝OV

琵

OI1 10DzK

的196bh ilと:ご=:≡

S211

&OEb

触れl山一DWh 2bEh蜘dI

ilr.1 i一｢

0■

11恥 0,6㌦

9Da

∝p ITIV Oll BOO htrLE

紗 SmS(Xil望

▲牡恥DtSm

CmOZm100 a pLnLPll

PZ; Zh

418B1 67)231

･873 627

●▲h:lP la)8加

7酬

OJMll

lB llBBl

Glucose twit

ODS Amide･80 MT

200.2

200.3

G叫114GIcNAc p l･2 Mana1‑6

MaTIPl･4GIcNAcβ1･4GIcNAc 9･4 GIcNAc B l･2 MaTtα1･3

GIcNAc B 1‑2Mand･6

ManP1‑4GIcNAcPll4GIcNAc 9･6 GAIPIJGIcNAc P l･2 Manα1･3

Ga岬114GLcNAc P 1‑2 ManoLl･6

MarLPll4GIcNAcP1‑4GJcNAc lO･2 Ga岬1‑4GIcNAc β I‑2 MancE1‑3

5.9 1 559.4

6. 1 1559.4

7.0 1 721.52

Figure II･32･ Structures of sugarchains corresponding to the number of 2･D map.

8 0 0 神仙軸心卸か LH HH Hu uH ra

整髪賢撃墜穿恥

Table III･l The positions of disulfide bonds of SML

Disu)ride bond Peptide fragment Ca)cuhted fragment peptjde mass

Ion mass MH+ (m/Z

1AVPTETTTTC DGNHVHR17

C(10)‑C(40) 35nEy,'sLELIc%LKT4Ulln YnK ,946･3 2947.5

C(20,･C(,,, ;:ksYciPcIFGIY.2㌦ 1377.6 .,,8.2

C(54,･C(S̀, :慧sQisp"C%cTSYt." 2356.0* 2,56.8

C(67)‑C(73) 60vDVLKSR(盟ELSTN78 2093.4 2092.9

C(108,･C(138, :::;TN慧慧蒜csYE141 3003･l ,0.1.8 C｡17,･C(195, :哉:pGsQt:IZ::‑' 1775･8 1775.0

C(152,‑C(182) :;三vDflGSrSvKGIIS:,｡̀ 1917.8 1,1,.4

｡ ‥i:‑l｡ ‥̀二 =‑=‑:‑‑‑‑≡こ二二三二三二二Il.;=‑A̲̲::̲̲ ,二三三̲∴こ̲:̲̲ ̲̲:̲I̲書こ̲

C(163)･C(169) 1855.0 1855.0

Underllne Onthe posidon Asp168 indlCateS the N‑llnked glycosyladon slte.

N C

Malls(邑N)

F i

g u

r e

t t

I 3

3 ･

M A

L D

t ･

T O

F m

a s

S s

p e

c t

r u

n o

f ‑

h e

g l

y c

o p

e p

t i

d e

r i

v e

d f

r o

m S

M L

.

賢

離

TをEC･

SI SK SK

丁【IllI

･lN*lT

‑‑EKSLT

‑AKXAVV

‑TIES

‑TRS

‑ll S IAtS IAIS

‑l SS

･l SS

‑l SS llYL

･KTS

‑KTS

一一VV11LE

‑‑YVIlLE

‑QVYHLE INVOFLl

･NVOFLl

･HVMRLS

･EIVOKLrl

‑FVORLS

GYAY ESAFI ESArl 8SAS SOAP SDAO SDAL

$OAL SDIL SDSO SDSCl SDDO SVAN

………;;‡………;

帯川一･･‑SOSVI KEFtV如D･=･SSLTl

≡…ri88'E::二:二手ぎT:と

二::…ヒ指

しflESEIVE

‑‑MNGETSTK

‑‑‑EIQRFtFL

･LISEl0RFtFL

‑NSEJFHRFL

一･･･TSIL EMTSSISL I‑‑‑･PSL

‑一日P‡L 一･･一･PSL

蓄

蕉き06RPOSO 監…:QB;;:…三 6‑DCY･YFLOKALYGFHDSq 6･OGY‑VFLOKALYGFHDSq Q‑OCV‑YFLOltALYOFITDSFI D‑SQY･lHERALYCFtTD(iT EI‑AQV･lHERALYGFLTDQT D･lGY‑lSVOTALYQFLEEISE EITBL一日VKSSLYOFtTDST D･SQV‑LNYNSITFQRTNSN DA･ lL=/NANYQltAS$01 4T･lOINTAHYBRTNKFT C卜IKYIKANYQRTDCKT

KT ‖l▲NYGFITD

tlK‑JOIXFtANY￠FHtQN GE･川lORAMYQFIFnN GE‑lR川L▲NYBFEROH GE‑日日QFuNY8FLFtOIl

NVFMANYQFZRDGS KYFHANYQFtRDCS

KYFHAJIYGMDQS

MYNHANYGRFtDLV HIHHANY(iRRDLY VIYDAlY8FIKRGE

lECKSLMHS STNfLPPAQVA SVeFtFIOtlOlY SNQLPNCLTO SEQFtPSSELO SBKFIATFElS

qSFKFIPANOLAN 萱…ほG:EE:冨LL;≡

挺;害RR,I:::LLKS:

瞳事t!蔓宅

蔓…三吉:二::ヒ;::

瞥;:::::‡E≡宇S' 替pClLFGAFT川牡sHIRPASOTAK

≡:;;:丁:冒王:Yy芸芸;::欄三g;E3::::

OYKlSL‖)lKYBLHNR‑‑一･一一1‑‑‑‑I KYTIFtYIEAtY(ilSQR‑‑･‑‑‑‑･･‑‑l GY‑川VYGVOFSTIトLSStltRP‑‑‑‑一

〇V‑lKVY(iYOrSTIEKLSSNEFW‑‑=‑

GV一日YYGYKFSTl卜KSSNESP‑‑一･

弼

Ot6‑TLILVISq OEC･TLALWSq OtQ･TLALYrS8 0T6‑TLEVLSH OT(卜TLtVL$O LLG‑AVDVLKS LP‑‑lTTldD LIV･‑YPEVSK

TEA‑SLS‖lSS

SOSJTTSKSAE SOSITTSKVAE SO‡‑STS川AE SOS‑STSKLllf SOS‑TTSKLJAE

I‑YNDG‑TSKVR lぜsDT‑EAKNGPS

膝ETT:…王E:冨ご;

EVl川RV･NQ EYKLIRV‑NQ EVNNRV‑NO tVNTEV‑FR EVNTEV‑FFL ELSTN卜FFt

描芸t;yD畳…t芸TT:と二LL王:

√､■7t?G.= R lq C'C'O∧u O G 0 t'一q CI 6 ら lq O Z hⅣ一q eI G C)6‑

沼櫛叩認蜘暇脚は聴峠聯縦軸蜘治榊脚脚将脚脚

VPAVN‑SVF YPAYN･SVF IPANN･IIFF VPASN‑SLY VPASN‑SLY

謁.;

#

PVFGD SYFSD

LDVAYTQLY一一‑H LEVAYV如ypsIV‑

LtVSYTtLGG!PTV 罪YGTYK‑I‑YLEVSYT果しGGSPTy 軒QTYK1‑‑YLEVSYTCLGGSPV‑

悪vv芸TH::::t…H

VASN‑lVFSDFI色VGTIK‑

VAGS‑DAFPEIPOPGTYK‑

VT6S‑DVFPDPg̲PCTYKI KASN‑8VFGpPYKCKKK‑

VITSGI VITS6‑

YLAVTY FLYVSY YLtVOY YL【VOY FtLLIKY DLFtVFH FtDVTY

Figure III･28･ Multiple alignment of the separated domain of rhamnose binding lectins.

Tandemly repeated domains of RBLsare separatelyaligned for comparison. Shadow boxes indicate con.served cysteine residues･ Underline indicates N‑linked glycosylation site･ Sequences were aligned with Clustal W and

alignment score was 124066･ SML: Spanish mackerel (Scomberomorous nl.Phonius) egg lectin･, STL: steelhead

trout (Oncorhynchus mykiss) egg lectin; CLS: chum salmon (Oncorhynchus keta) egg lectin; SAL: catfish (Silu,us asotus) egg lectin; WCL: white‑spotted charr (SaLvelinus leucomaenis) egg lectin; TBL: fareastem dace

(Tribolodon taczanowskii) egg lectin; SUEL: sea urchin (Anthocidaris crassispina) egg lectin; PPL: mantle of

penguin wing oyster (Pteria Penguin) lectin; LPH: α llatrotoxin recepter from rat (Rattus nonJegicus); LPH2: α ‑

latrotoxin recepter from human.

Fig.10

Order

Adyanced species

Figure IV･1. Hierarchy of higher categories of fishes.

Bold letters indicatethe fish species, which contain rhamnose‑binding lectins in eggs.

棚帥

㈹㈹

ドキュメント内魚病細菌・原虫に結合する魚類レクチン・ファミリーの生体防御機能 (ページ 79-89)

Fig. 8

Fig. 9

Figure II･14･ HPLC chromatogram of PA･glucose oligomers (DP =

( % ) T O u q n 甲 T

( % ) t o u q q Z ･ 1

Figure II‑18. HPLC chJ.OmatOgram Of PA･sugar chain 009 0n a

0 D S ･ s i l i c a ( g l u c o s e u m i t s ) F i

g u r e t t

･ ) 9 ･ T w o J i m e n s i o n a l m a p o f P A t a g g e d s u g a r c b a i n s

･

GIucose unit

001

100.1

210.4

GaLPl｡G.cNAcPL2Manα1.TanP1‑4GIc"AcβL4GIc"Ac 13･8 7･4 1883･72

Figure II122･ Structures of sugar chains corresponding to the number of 2･D map･

蜘 叫

仙 帆

. W

m 温

伽

Figtlre I1‑27･ HPLC of PAISugarChains derived from SML on a

TSKge1 0DS 120T column (4.6 X 250 Jnm).

3 3 t Z 3 3 S a

･ 1 0 n T a

( % ) t o t r q q Z

･ 1

3 3 u 3 3 S 3 J O n t A

( % ) T O t Z q n t [ ･ T

Figure II‑29･ HPLC ofsugarchains derived from SML ob a TSKgel

Mr, 172132

Figure II･31･ MALDI･TOF mass spectrum of PAISugar Ctlains

器

PZ; Zh

7酬

Figure II･32･ Structures of sugarchains corresponding to the number of 2･D map.

整髪賢撃墜穿恥

Disu)ride bond Peptide fragment Ca)cuhted fragment peptjde mass

1AVPTETTTTC DGNHVHR17

C(54,･C(S̀, :慧sQisp"C%cTSYt." 2356.0* 2,56.8

C(108,･C(138, :::;TN慧慧蒜csYE141 3003･l ,0.1.8 C｡17,･C(195, :哉:pGsQt:IZ::‑' 1775･8 1775.0

C(152,‑C(182) :;三vDflGSrSvKGIIS:,｡̀ 1917.8 1,1,.4

F i

g u

r e

t t

I 3

3 ･

M A

L D

t ･

T O

F m

a s

S s

p e

c t

r u

n o

f ‑

h e

g l

y c

o p

e p

t i

d e

d e

r i

v e

d f

r o

m S

M L

.

賢

離

………;;‡………;

二::…ヒ指

蜘叫

仙帆

棚帥

㈹㈹