Hypervariable regions in the putative glycoprotein of hepatitis C virus

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 175, No. 1, 1991

Pages 220-228

February 28, 1991

HYPERVARIABLE

R E G I O N S IN THE P U T A T I V E HEPATITIS C VIRUS I

GLYCOPROTEIN

OF

M a k o t o Hijikata, N o b u y u k i Kato, Yuko Ootsuyama, M a s a n o r i Nakagawa, Shogo Ohkoshi, and K u n i t a d a S h i m o t o h n o

Virology

Division, N a t i o n a l Cancer C e n t e r R e s e a r c h Institute, 5-1-1, Tsukiji, Chuo-ku, T o k y o 104, Japan

Received January ii, 1991 SUMMARY: A c o m p a r i s o n of the s e q u e n c e s of the p u t a t i v e g l y c o p r o t e i n r e g i o n in t h r e e i n d e p e n d e n t c D N A clones of h e p a t i t i s C virus a n d of s e q u e n c e s of four o t h e r clones revealed extensive g e n e t i c v a r i a t i o n c l u s t e r e d and i n t e r s p e r s e d w i t h h i g h l y c o n s e r v e d a m i n o a c i d sequences. We o b t a i n e d e v i d e n c e for two h y p e r v a r i a b l e r e g i o n s in the p u t a t i v e e n v e l o p e g l y c o p r o t e i n , one r e g i o n was a s s u m e d to be a p o t e n t i a l a n t i g e n i c site, as d e d u c e d f r o m the h y d r o p h i l i c i t y and a n a l y s e s of s e c o n d a r y s t r u c t u r e s . These o b s e r v a t i o n s suggest the e x i s t e n c e of a large pool of a n t i g e n i c v a r i a n t s of h e p a t i t i s C virus, in Japan. ©1991AcademicPress,Inc.

Hepatitis molecularly agents

C virus

cloned

(HCV),

(i, 2),

of p o s t - t r a n s f u s i o n

apparently

an e n v e l o p e d

RNA genome

encoding

the g e n o m e

is t h o u g h t non-A,

virus

non-B,

carmovirus

deduced viral

from

a large p o l y p r o t e i n

structural

glycoprotein(s)

protein

United amino

of the

(HCV-US)

acid positions

difference) (2) . putative

envelope

c D N A clones

of p l a n t

encoding

region

and in Japan results

as

in amino

precursor

(HCV-J) (35% d i f f e r e n c e ) , conserved

For purposes

the a m i n o - t e r m i n a l

portion

(2).

acid positions

the H C V g e n o m e s

relatively

a

the e n v e l o p e

suggested polymorphism

of HCV.

as well

Although

to locate

from amino

diverged between

protein(s)

This virus

viruses,

(4).

identified,

estimated

500 to 750 were

These

H C V is

stranded

(i) .

190 to 700 on the v i r a l p o l y p r o t e i n

acid sequence

States

precursor

similarities

has not b e e n

350 to 500 was e x t e n s i v e l y

(3).

single

and to f l a v i v i r u s e s ,

families

acid sequence

of H C V was r o u g h l y

acid positions The a m i n o

and p o t y v i r u s

amino

hepatitis

with a positive,

seems to be r e l a t e d to p e s t i v i r u s e s as the

of w h i c h has b e e n

to be one of the c a u s a t i v e

in the while

(16%

of the

of e l u c i d a t i o n ,

of the p u t a t i v e

1 The n u c l e o t i d e s e q u e n c e s of clones 63, 64, a n d 168 have b e e n s u b m i t t e d to the D D B J / E M B L / G e n B a n k D N A d a t a b a s e s w i t h a c c e s s i o n number(s) D00689, D00690, a n d D00691, r e s p e c t i v e l y . 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction #z any form reserved.

220

Vol. 175, No. 1, 1991

envelope of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

protein

9 Japanese

sequenced,

from a e D N A l i b r a r y

patients

originating

with PT-NANBH

and compared.

6) w e r e a l s o e x a m i n e d

Four

for p u r p o s e s

MATERIALS

(2), were

sequences

from plasma

RNAs

screened,

already

reported

(2, 5,

of c o m p a r i s o n .

AND M E T H O D S

¢ i Q n i n g of ¢ D N A s e q u @ n c e s ¢ Q n t a i n i n a %h@ H G V s t r u c t u r a l P r o t e i n reQion. A k g t l l c D N A library was c o n s t r u c t e d f r o m p l a s m a RNAs from 9 p a t i e n t s w i t h NANBH, u s i n g a H C V s p e c i f i c primer, 5'GGGCTCGGAGTGAAGCAATA-3' (nucleotide p o s i t i o n s 1848 to 1867 in H C V genome) (2) . The c D N A l i b r a r y was s c r e e n e d u s i n g as a p r o b e a fragment of clone 4 (nucleotide p o s i t i o n 1718 to 2098 in H C V genome) (2) . A f t e r t e r t i a r y screening, p o s i t i v e p l a q u e s w e r e p u r i f i e d and c D N A inserts of these clones were p u r i f i e d a n d s u b c l o n e d into the p T Z I 9 R v e c t o r (United States B i o c h e m i c a l Corp.). D N A s e q u e n c e analysis. R e c o m b i n a n t s were s e q u e n c e d as d o u b l e - s t r a n d e d D N A b y the d i d e o x y chain t e r m i n a t i o n m e t h o d (7), u s i n g S e q u e n a s e D N A s e q u e n c i n g kits (United States B i o c h e m i c a l Corp.). Sets of 5' a n d 3' d e l e t i o n s of the inserts were p r e p a r e d u s i n g e x o n u c l e a s e III (Takara Shuzo) for s e q u e n c i n g (8). P r o t e i n analysis. H y d r o p h i l i c i t y a n a l y s i s of the d e d u c e d a m i n o a c i d s e q u e n c e s of c l o n e d c D N A clones was p e r f o r m e d a c c o r d i n g to H o p p a n d W o o d s (9), u s i n g a w i n d o w of 7 c o n s e c u t i v e residues. The s e c o n d a r y s t r u c t u r e s of the p r o t e i n s were a n a l y s e d a c c o r d i n g to Chou a n d F a s m a n (I0) .

RESULTS Five finally 171 were 64,

c D N A clones,

obtained.

n a m e d clone

Since

identical

and 168 were

AND D I S C U S S I O N 63,

nucleotide

to clones

selected

64,

158,

sequences

63 and 3

(2),

for f u r t h e r

168,

and 171,

of clones

respectively,

analyses.

clone

Comparisons

the n u c l e o t i d e

and amino a c i d s e q u e n c e s

of the 3 i s o l a t e d

as well

4 reported

(2, 5,

as the

Fig.

i.

Table

i.

The d i f f e r e n c e s

These

7 clones

nucleotides and

63

I).

differed

Clone

following

reasons:

5

(NS5)

between

H C V genomes,

region

The region

region

(2),

showed

in H C V - J clones variation

sequence

Clones

between

3 in o n l y

PT-NANBH,

of the p u t a t i v e

of the p u t a t i v e

f r o m a single p a t i e n t

221

in

of

clone

3

1.6% of

for the regions

less than a 2.5% n u c l e o t i d e

data).

shown

non-structural

conserved

f r o m 19 i n d e p e n d e n t

(unpublished

in

63 and 3 are l i k e l y to

with

from 1.0% to 2.0% d i f f e r e n c e s , level

except

one of the m o s t

in p a r t

of H C V - J g e n o m e s

the range

acids).

clones,

in 8.3 to 30.4%

f r o m clone

f r o m a single p a t i e n t

protein

cases,

of amino acids),

are

63,

of

6), are shown

acid sequences

from each other

63 d i f f e r e d

(3.2% of amino

have o r i g i n a t e d

diversity

in the amino

(8.4 to 27.9%

(Fig.

nucleotides

many

HCV sequences

were

158 and

patients

sequence (ii) .

with PT-NANBH

at the n u c l e o t i d e

However,

In

non-structural

it cannot

be

is in

Vol. 175, No. 1, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HCJI C A TCCA A C T T C T T C T G C GCAC TGCC C T T G T YEK T A A C G G TG HCJ4 T A C T T 168 T A G C AT G T T 64 T A C A GT T 63 3 TACGAGGTGCGCAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGTATTGTGTATGAGGCAGCGGACATGATCATGCACACCCCCGGGTGC i007 3 Y E V R N V S G I Y H V T N D C S N S S I V Y Z A A E M I M H T P G C 226 63 64 T S 168 V D V HCJ4 YEK V A HCJI Q S T L P H A L

HCJI C T T C G C CG GAG T G A G C G A CA G G AAC G G CAGC T T YEK T AC C TGC G T T HCJ4 T GAC CAG TGC G T 168 T AGCCG CA C G T C G T 64 T GAC C G TGC G T T T 63 3 GTGCCCTGCGTCCGGGAGAGTAATTTCTCCCGTTGCTGGGTAGCGCTCACTCCCACGCTCGCGGCCAGGAACAGCAGCATCCCCACCACGACAATACGACGCCAC 3 V P ~ V R E S N F S R ~ W V A L T P T L A A R N S S I P T T T I R R H 63 64 D S A V 168 A D H T V HCJ4 D S A V YEK N S A V L HCJI G V M V T D G K L A Q L

1112 261

HCJI A C T C AGC CA C G CC C G G G C C TA TGGT A T T CA G YEK C A T C G T C A G HCJ4 C T C G T C G 168 C T C G T G 64 C T G T T C A G 63 T 3 GTCGATTTGCTCGTTGGGGCGGCTGCTCTcTGTTCCGCTATGTACGTTGGGGATCTCTGCGGATCCGTTTTTCTCGTCTCCCAGCTGTTCACCTTCTCACCTCGC 1217

3 V D L L V G A A A L ~ S A M Y V G D L ~ G S V F L V S Q L F T F S P R 2 9 6 63 64 168 HCJ4 YEK HCJI

T S

I

F F F F

I

T

I I

L

G

HCJI CC CTG AACG GC T T C TA A G T A C GG G YEK C A G C C C T G C GG A HCJ4 C A G G C C TT A 168 C AAC G C C C T G A 64 C A G C C A T GG A 63 3 CGGTATGAGACGGTACAAGATTGCAATTGCTCAATCTATCCCGGCCACGTATCAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCACCTACAACGGCC 1322 3 R Y E T V Q E ~ N --C S I Y P G H V S G H R M A W D M M M N W S P T T A 331 63 64 H L T A 168 H T L A HCJ4 H L YEK H A HCJI H W T G L T A

HCJI T G A A GG T G A T T A C T T T A G A A G T TC YEK T G T T A A HCJ4 G T G T A G A 168 G T T A G A 64 G T T A T A G T 63 3 CTAGTGGTATCGCAGCTACTCcGGATCCCAcAAGCCGTcGTGGACATGGTGGcGGGGGCCCACTGGGGTGTCCTAGCGGG•CTTGCCTACTATTCCATGGTGGGG 3 L V V S Q L L R I P Q A V V D M V A G A H W G V L A G L A Y Y S M V G 63 64 168 HCJ4 YEK HCJI

M MA

IL

I

I

F

Fig. i. A l i g n m e n t of n u c l e o t i d e and a m i n o a c i d s e q u e n c e s ( s i n g l e - l e t t e r code) w i t h i n the p u t a t i v e e n v e l o p e r e g i o n of 7 H C V g e n o m e s (clone 3 (2), 63, 64, 168, HCJI (6), HCJ4 (6), a n d t e n t a t i v e l y n a m e d YEK (5)). S e q u e n c e of clone 3 is u s e d as the reference, c D N A a n d a m i n o a c i d s e q u e n c e s are a l i g n e d a b o v e and b e l o w the reference, r e s p e c t i v e l y . N u c l e o t i d e s a n d a m i n o acids are a c c o r d i n g to the e n t i r e s e q u e n c e of the H C V - J g e n o m e (2). O n l y d i f f e r e n t r e s i d u e s from the r e f e r e n c e s e q u e n c e are shown. R e p o r t e d range of the YEK s e q u e n c e in this r e g i o n is i n d i c a t e d in parenthesis. P o t e n t i a l N - g l y c o s y l a t i o n sites (N-X-S/T) are underlined. I n v a r i e d 14 c y s t e i n e r e s i d u e s are d o u b l e - u n d e r l i n e d . H y p e r v a r i a b l e r e g i o n s 1 and 2 are boxed.

222

1427 366

Vol. 175, No. 1, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HCJI G C GA C TG G C C CGA AT CTC C~CC CGGC TGTCTGA T TAGT YEK T C T G G GGT CA G C A GT TGT CA AC HCJ4 C GC C C G G T ACT G GGC CCAG CA C TCC CG C 168 TC C G C C AG G TG GGCA CCAG AG C T C GT T C 64 T C G A G G CGGT CA G CA G G TCG G C 63 G CA T A CG 3 ~CTGGGCT~GGTCTTGATTGT~TGCTACTCTTT~TGGCGTTGACGG~ACACCCACGTGACAGGGGG~GGGTAGCCTCCAGCACCCA~GCCTCGTGTCC 1532 3 N W A K V L I V M L L F A G V D G H T H V T G G R V A S S T Q S L V S 4 0 1 63 R Q F R H G 64 E R A V Q G H G A L G A 168 E M A A S T Y R F A HCJ4 A E Y T S A A S H T ST A YEK R V Q G H V CT T HCJI V L A E I S Q A R A M S G

HCJI YEK HCJ4 168 64 63 3 3 63 64 168 HCJ4 YEE HCJ]

CTCT A C G TAAG C G GA C T T C G CT C AAG TA C C C CTCT TAG CT GG G C T G T A T T T T C C CTCT CT GG G G G T T T C A C C CTCT A TCT G GG G T A T T CT C G CTCT TA CT G GAGC G TA A T T C C C A G T T A G TGGCTCTCACAAGGCCCATCTCAGAAAATCCAACTCGTGAACACCAACGGCAGCTGGCACATCAACAGGACCGCTCTGAATTGCAATGACTCCCTCCAAACTGGG 1637 W L S Q G P S Q K I Q L V N T N G S W H I N R T A L N ~ N D S L Q T G 436 L F T L F T P I E L F T S A L F P A R H L F R P A L F T P A K N I S E N

HCJI GGT A A G TA TCA AA T T G C T C A GT C A G C TACC T T AC C C T YEK C C ) HCJ4 C C A T G C T CTG C A C 168 TC C G AT T T A TC C T TT CA AG A 64 GGC C C TA AG AA A G C T T CAC A A C 63 C 3 TTCATTGCTGCGCTGTTCTACGCACACAGGTTCAACGCGTCCGGGTGCCCAGAGCGCATGGCTAGCTGCCGCCCCATCGATGAGTTCGCTCAGGGGTGGGGTCCC 1742 3 F I A A L F Y A H R F N. . . A. . . S G =C P E R M A S C R P I D E F A Q G W G P 471 63 64 W L T S K S T D 168 R N S Q S K E HCJ4 L T S W YEK L ) HCJI W L G I Q K S L R L T D D

HCJI C CC A C G G A A G C G C C AC C C T C C AAA T T C AAA AGC A C G A HCJ4 CT AC GA C C T T T T T A C 168 T C GA AG GCG C T TA A TA C T C G 64 T TC A T A CG A G T A A T TG T A A C 63 TG 3 ATCACTCATGATATGCCTGAGAGCTCGGACCAGAGGCCATATTGCTGGCACTACGCGCCTCGACCGTGCGGGATCGTGCCTGCGTCGCAGGTGTGTGGTCCAGTG 1847 3 I T H D M P E S S D Q R P Y ~ W H Y A P R P ~ G I V P A S Q V ~ G P V 506 63 A 64 Y V K S N G E Q V I 168 Y A E Q G G P N Q S E HCJ4 Y T E D P HCJI S A N G S G P P K K S

Fig. 1-Continued

excluded

that the h i g h l y h o m o l o g o u s

patients

w i t h NANBH.

of a m i n o

acid difference

i) .

This

diversity

the HCV-US, with

On the other hand, (26.4%)

between

of HCJI p r o b a b l y

as s u g g e s t e d

imported

HCV clones

clotting

(2, 6).

factors

there HCJI

means

Japanese

have

infected

some

was a h i g h a v e r a g e and others

(Table

that HCJI b e l o n g s hemophiliacs

m a y have b e e n

infected

to

transfused

with HCV-US

(12). Extensive HCV isolates (13,

14).

About

is a p p a r e n t

Differences

nucleotide contrast

variation

changes

in p u t a t i v e

genes

42% of the n u c l e o t i d e

substitutions,

glycoproteins

a n d like that of H I V e n v e l o p e

a m o n g the 7 s e q u e n c e s

and insertion

to e n v e l o p e

envelope

appeared

and d e l e t i o n

of the H I V isolates changes

lead to a m i n o

the result b e i n g n u m e r o u s

223

amino

proteins as single

were absent, (13,

of

in

14) (Fig.

I) .

acid

acid variations.

Vol. 175, No. 1, 1991

Table I.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Differences in amino acid sequences in the putative envelope region between seven

clone

region b

3

63

64

168

HCJ4

YEK

HCV genomes a

63

64

168

HCJ4

YEK c

HCJI

1

i0/ 3.2 d

49/15.6

47/14.9

35/11.I

28/11.2

88/27.9

2

5/50.0

8/80.0

8/80.0

8/80.0

8/80.0

7/70.0

3

0/0

6/85.7

7/100.0

5/71.4

6/85.7

1

45/14.3

47/14.9

35/11.1

25/10.0

84/26.7

2

8/80.0

8/80.0

8/80.0

8/80.0

5/50.0

3

6/58.7

7/100.0

5/71.4

6/85.7

1

49/15.6

41/13.0

21/ 8.4

82/26.0

2

9/90.0

7/70.0

5/50.0

8/80.0

3

5/71.4

6/85.7

6/85.7

1

40/12.7

29/11.6

87/27.6

2

4/40.0

9/90.0

9/90.0

3

4/57.1

4/57.1

1

23/ 9.2

79/25.1

2

6/60.0

7/70.0

3

6/58.7

1

62/24.8

2

9/90.0

a. Based on the aligned amino acid sequences shown in Fig.l. b. i. Entire sequence (315 amino acids, amino acid positions from 192 to 506). 2. Hypervariable region 1 (i0 amino acids, amino acid positions from 391 to 400). 3. Hypervariable region 2 (7 amino acids, amino acid positions 474 to 480). c. Reported amino acid sequences of YEK covers 250 amino acids in the region 1 (amino acid positions from 192 to 471) and does not contain the hypervariable region 2, as shown in Fig. I. d. Number of amino acid differences/% diversity of amino acid sequence.

However,

despite

distribution region amino

which acid

14) (Fig.

the

in t h e is

extensive is not

clustered

sequences 2).

overall variation

as

and

seen

In p a r t i c u l a r ,

sequence

uniform

interspersed in t h e

regions

from

391

to

400

(hypervariable

region

i)

and

474

to

480

(hypervariable

region

2)

showed

224

with

envelope

variation,

throughout highly

genes

amino

from

this

acid

amino

a 40.0

of

conserved HIV

positions

acid to

(13,

positions

90.0%

and

Vol. 175, No. 1, 1991

57.1

to

100.0%

except

i,

in

although

clone

were region 14

of

invariant

These

structure The a result the

o-

the

of

(Table

was

(Table

i).

envelope

were may

in t h e

and

a large was most

in t h e s e

among

viral

replication

errors

and

This

raises RNA

highly of

is

the

HCV

by

of

each

the

potential regions

N(Fig.

a higher-order isolates.

a

possibility

polymerase

of

probably

followed the

the

of

sequences

region

2 of

conserved,

conserved

conservation

glycoproteins

RNA-dependent

hypervariable

part

facilitate

of

clones, a 35.3%

However,

residues

nature

here,

region

glycoprotein

located

7 HCV

Even

in h y p e r v a r i a b l e

population. the

between i) .

observed

sequences

envelope

polymorphic of

acids

63

cysteine

sites

of

respectively,

3 and

acid

features

mutated

fidelity

amino

identical

glycosylation 2).

clone

amino

other and

diversity,

between

difference

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HCV,

arises

selection that

as of

the

possibly

YEVRNVSG YHVTND~SNSSIVYEA D IMHTPG~VPIVRE ~__$_R=CWVALTPTLAARN PTTT RRHVDLLVG AA

271

U Z

H

0

~ ~SAMYVGDL~GSVFL SQLFTFSPRR ET QD~N~SIYPGH GHRMAWDMMMNWSPT ALVVSQLLRIPQAV DMVAGA 351

H

HWGVLAGLAYYSMVGNWAKVLIV LLFAGVDG T V GG["--'----~SLF G Q IQL NTN_~WHI~_I~ALNCN 431

,A S L TG AALFY

,A

FN SG~PERMAS~_R ID F QGWGPIT[-----~DQRPY~WHYAPR ~GIVPAS V~GPV

Fig. 2. Schematic d i a g r a m of relative variation, or d i v e r g e n c e in the p u t a t i v e envelope region of 7 HCV-J clones. Relative degrees of v a r i a t i o n were c a l c u l a t e d and p l o t t e d on the ordinate from 0 (minimum divergence) to i0 (maximum divergence) (13) . Single letters indicate the c o n s e r v e d amino acids, d e f i n e d by identity in seven out of seven or six out of seven of the sequences shown b e n e a t h the abscissa. The amino acid p o s i t i o n number is i n d i c a t e d in accordance with the entire sequence of HCV-J genome (2). Invaried p o t e n t i a l N - g l y c o s y l a t i o n sites and 14 cysteine residues are u n d e r l i n e d and double-underlined, respectively. H y p e r v a r i a b l e regions 1 and 2 are boxed.

225

506

Vol. 175, No. 1, 1991

encoded the

in t h e N S 5

reverse

probable serves visna (20,

21),

case

of H I V

residues

was

antigenic

(17,

18),

and/or

no

24,

the

way

that

respectively

criteria

with

highly glycoprotein

as

indicated virus

for t h e

(19),

tropism,

and HIV

as

in the

for t h e p e r s i s t e n t

these

highly

cannot

region

variable

be e x c l u d e d .

2 of all H C V

and had a predicted analysis

case

envelope

in t h e v i r a l

make

hypervariable

(i0),

meet

is the

It s e e m s

anemia

importance

hydrophilic

analysis

~ turn

(9) a n d p r o t e i n (Fig.

for p r o b a b l e

3).

The

clones

structure, secondary

These

antigenic

epitopes

(23,

25). As t h e r e

region, HCV

changes

in a h y d r o p h i l i c i t y

characteristics

of HCV,

The p o s s i b i l i t y

surrounding

as

16).

infectious

of w h i c h

functional

extensively

(15,

low,

of p u t a t i v e

variation

equine

both

of HCV.

structure

is q u i t e

of H I V

may cause

(22),

bear

as n o t e d

(2),

the heterogeneity

as the

infection

region

transcriptase

that

virus

region

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the possibility

in J a p a n

developing account

is s e q u e n c e

effective

would

of t h i s

envelope

of a n t i g e n i c

variants

n e e d to be c o n s i d e r e d .

therapeutic

be t a k e n

in the p u t a t i v e

of a l a r g e p o o l

and elsewhere

must

~;

variability

strategies

and related

for H C V

of

When

infection,

factors.

clone63

2

TT

T

T

T

;

TTT

~-2 1 - 3

. . . .

l

250

x 0>

clone3

-

1

t't1"

~O ~ - 2 I HVR2 "~1

~',rr~2

~wi I

200

• ,-I

T

- 3 J .

~ 460

.

. . . .

i

300 clone64

. . . .

I

i

. . . .

400

clone168

' HVR2

. . . .

350

I

. . . .

450

l

'

500 a.a.

HCJ4

HCJI

HVR2

HVR2

HVR2

.

490

460 '4'9b 460 "4"96 460 "4'90 460 '4'90 ....

Fia. 3. Structural features of the putative envelope proteins of six HCV clones. Hydrophilicity profile of the protein of clone 63 is shown. Results of the region from residues 192 to 510 of clone 63 and regions surrounding HVR2 (residues 460 to 490) of clone 3, 64, 168, HCJ4, and HCJI are also shown. Hydrophilicity profiles of each clone showed a similar pattern. Locations of predicted ~ turn structures are indicated by asterisks. Potential N-glycosylation sites are indicated by arrowheads. Hypervariable region 1 (HVRI) and 2 (HVR2) are indicated by thick bars.

226

Vol. 175, No. 1, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS: We thank M. Ohara for comments. This work was supported in part by a Grant in-Aid from the Ministry of Health and Welfare for a Comprehensive 10-Year Strategy for Cancer Control and a Grant in-Aid from the Ministry of Science and Culture, Japan. M.H. is a receipient of a Research Resident fellowship from the Foundation for Promotion of Cancer Research, Japan.

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