Eggshell precursor proteins of Fasciola hepatica, II. Microheterogeneity in vitelline protein B

Molecular and Biochemical Parasitology, 54 (1992) 143 152

143

~:3 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01787

Eggshell precursor proteins of Fasciola hepatica, II. Microheterogeneity in vitelline protein B J. H e r b e r t W a i t e a a n d Allison C. R i c e - F i c h t b aMarine Biology/Biochemistry Program, College of Marine Studies, University of Delaware, Lewes, DE, USA, and bDepartment of Medical Biochemistry and Genetics, Texas A & M University, College Station, TX, USA (Received 21 November 1991; accepted 14 February 1992)

At least 3 structural protein precursors of the eggshell are synthesized and stockpiled in the extensive vitelline cells of the liver fluke Fasciola hepatica L. One of these, vitelline protein B, consists of a closely related family of proteins that owes its apparent electrophoretic heterogeneity to variations in the Tyr to DOPA conversion as well as to subtle variations in the primary sequence. The efficiency of the Tyr to DOPA conversion ranges from a maximum of about 90% to a minimum of 55% in the protein. Trypsin digestion in borate buffer at pH 8 was used to produce DOPA-peptides for sequencing. Notably, trypsin does not cleave Arg/Lys-DOPA sequences at borate concentrations greater than 0.15 M. Peptides with DOPAcontaining sequences most frequently have flanking amino acids such as Lys, Ser, or Asp on the N-terminal side and Gly or Asp on the C-terminal side. All protein variants fall within a narrow molecular weight range (30 33 kDa), a pl range of 6.9 to 8.3, and the collective majority would appear to share a common N-terminal sequence up to residue 28. The results suggest some combination of the following: variations in post-translational hydroxylation, alternative post-transcriptional splicing and/or the existence of multiple gene copies of eggshell precursors. The latter have been shown to occur in the blood fluke Schistosoma mansoni [15]. Key words: Fasciola hepatica; Vitelline protein; Eggshell precursor; Microheterogeneity; DOPA-peptide

Introduction

Eggshell formation is a crucial step in the reproduction of trematodes. Without eggshells, embryonic trematodes would be exposed to the host organism's immune defenses and digestive enzymes, as well as environmental adversities once released. Eggshell formation appears to involve an orderly assembly of soluble eggshell precursors followed by a chemical crossCorrespondence address." J. Herbert Waite, Marine Biology/ Biochemistry Program, College of Marine Studies, University of Delaware, Lewes, DE 19958, USA. Tel.: (302) 645-4257; Fax: (302) 645-4007. Abbreviations: SDS, so~lium dodecyl sulfate; DOPA, 3,4dihydroxyphenyl-L-alanine; PTH, phenylthiohydantoin; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; vpB, viteUine protein B.

linking process known as quinone-tanning [1,2]. Cross-links have yet to be identified but may be derived from 3,4-dihydroxyphenylalanine (DOPA) residues in the precursor proteins following their oxidation to quinones by catechol oxidase [3]. Three DOPA-containing precursors have been identified from the vitellarium of the liver fluke Fasciola hepatica. These are referred to as vitelline proteins (vp) A, B, and C, and have apparent sizes of 70, 31, and 17 kDa, respectively [4]. That at least one of these (vpB) is extractable from eggshells released by F. hepatica in vitro is suggested by the detection of a 30-kDa protein in immunoblots screened with polyclonal antiserum prepared to a fusion protein derived from a female genital complex c D N A library of the worm [5]. All 3 vitelline proteins exhibit microheterogeneity following polyacryl-

144

amide gel electrophoresis. In vpC, however, only one species is visible by electrophoresis in acetic acid/urea and Tris-glycine with sodium dodecyl sulfate, whereas 2 are detectable by isoelectric focusing, and as many as 6 appear following electrophoresis in Tris-borate with sodium dodecyl sulfate [4]. The factors contributing to microheterogeneity are not presently understood and require further clarification. Three plausible causes of heterogeneity are variations in (i) primary sequence, (ii) molecular weight, and (iii) the efficiency of the post-translational modification of tyrosine to DOPA. In vpB, protein heterogeneity was not initially observed, in part, because of inadequate electrophoretic challenge of purity [6]. Following our experience with vpC, we have decided to expand our investigation of the primary structure of vpB as well. Our results suggest that microheterogeneity is caused by variations in sequence as well as in post-translational modification.

Materials and Methods

Extraction and purification of vitelline protein B. Liver flukes were obtained from condemned fresh bovine livers at a slaughterhouse (Braunfels Meats, Inc.) in Sealy, TX. After rinsing briefly with buffered 0.9 % NaC1, flukes were frozen in lots of 30 in liquid nitrogen. For dissection of the vitellaria, the flukes were prepared as detailed by Waite and Rice-Ficht [4]. In bulk preparations, the vitellaria were homogenized with chilled hand-held ground glass tissue grinders (Kontes, Vineland, N J) in 0.15 M NaC1 with 0.05 M Tris, pH 7.5, 1 m M phenylmethylsulfonyl fluoride, 10 m M N-ethylmaleimide, 25 m M E D T A and 1 m M potassium cyanide. Homogenates were centrifuged at 5000 x g for 10 min, and pellets were rehomogenized in cold 5% acetic acid with 4 M urea, 1 m M iodoacetamide and 10 /~M leupeptin (Boehringer Mannheim, Indianapolis, IN), followed by centrifugation for 20 min at 35000 x g. Supernatants were frozen at - 2 0 ° C until further use. When 100-200 ml of acid-

extracted supernatant had been accumulated, these extracts were thawed, brought to 10% (w/v) ammonium sulfate, then stirred vigorously for 60 min at 5°C and centrifuged as before to remove the precipitate. The second supernatant was collected and dialyzed overnight against 100-200 vols. of cold 2.5% acetic acid using dialysis tubing with a 1000-Da cutoff (Spectrum Industries, Los Angeles, CA). Dialysis produces a flocculum harvestable by centrifugation at 10000 x g for 30 min. Approximately 90% of the pellet consists of 2 proteins, i.e., vpB and vpC [4]. These proteins are easily redissolved in 5% acetic acid with 8 M urea, and purified by C-8 reversed phase (Aquapore RP-300 Brownlee) high performance liquid chromatography (HPLC) using a gradient of acetonitrile (0-30%) in double distilled water with 13 mM trifluoroacetic acid (TFA) at a flow rate of 1 ml/min -1. All fractions were lyophilized at - 8 0 ° C for 12 h. Proteins were hydrolysed in one of two ways: (A) 7 M HC1 with 10% TFA and 10% phenol in vacuo at 155°C for 22, 44, and 66 rain [7], or (B) 4 N methanesulfonic acid in vacuo at l l0°C for 24 h for the determination of tryptophan [8]. Amino acid analysis was routinely performed on a Beckman System 6300 Autoanalyzer (Beckman Instruments, Palo Alto, CA) using the ninhydrin reaction for detection.

Electrophoresis. Routine electrophoresis was done on polyacrylamide gels (7% acrylamide and 0.35% methylene-bis-acrylamide) containing 5% acetic acid and 8 M urea. Acid-urea electrophoresis requires a 2-3-h pre-equilibration [9], but it is convenient here because it lends itself to rapid staining for either protein or DOPA. Proteins were stained using 0.001% Serva Blue R in 7.5% acetic acid and 40% methanol, whilst DOPA was stained by the Arnow method [10]. Apparent molecular weights were determined by SDS polyacrylamide gel electrophoresis using either the discontinuous Tris-glycine [11] or Tris-borate buffer system [4,12]. For the latter, the polyacrylamide gels contained 10% acrylamide, 2.5% bis and 4 M urea whereas the

145

running buffer (gel and reservoir) contained 90 mM Tris/90 mM boric acid/2.5 mM EDTA (disodium)/0.1% SDS at a pH of 8.2. Isoelectric focusing of denatured proteins was performed using polyacrylamide in vertical gels with dimensions 90 x 90 x 0.75 mm [11]. Ampholytes used were pH range 3-10 (Pharmalytes, Pharmacia, Piscataway, N J) and pH ranges 7-9 and 8 10 (Pharmalytes, Pharmacia). Pre-equilibration conditions were 200 V for 30 min, 550 V for 30 min, and 1000 V for 1 h. Focusing was carried out at 500 V for 1 h and 1000 V for 2 h. The pH gradient was determined by measuring the pH of 5 mm slices and by the following standards: horse heart myoglobin (pI 6.76 and 7.16), and rabbit muscle lactate dehydrogenase (8.55) both from Sigma.

of DOPA peptides. Trypsin (Boehringer-Mannheim) digestion of proteins was carried out for 24 h at a protein to enzyme weight ratio of 50:1 in 0.05 or 0.15 M sodium borate, pH 8.2 with 3.5 M urea in ReactiVials (Pierce Chemicals) under constant stirring. The borate is absolutely essential to prevent

precipitation of vpB. The digestion was stopped by the addition of one tenth part of glacial acetic acid. Resultant peptides were purified by reversed phase HPLC using a C-8 column (250 x 4.5 mm, Brownlee RP-300) with a linear gradient of 0-9% acetonitrile or a C-18 column (250 x 4.6 ram, Microsorb, Rainin) with a gradient of (~25% acetonitrile.

Peptide and protein sequencing. Peptides and proteins were sequenced on a microsequenator from Porton Instruments (Tarzana, CA) using automated Edman chemistry. PTH-amino acids were identified by HPLC using a C-18 column (HP-79918 AA) and a gradient recommended by the manufacturer. PTHDOPA and PTH-Ala tend to coelute in this system, but are resolvable by modifications in the elution gradient of acetonitrile [13].

Preparation

vpc

VpB

;--,

;

o

,o

20

3o

4o'

Elution time

o'6o'z'o

',

°-2 .q-

;o

(min)

Fig. 1. Purification of vpB using reversed phase high performance liquid chromatography. Acetonitrile begins at 0%, inereases to 16% in 16 min, and then more gradually to 37% at 60 min; at 6 ~ 7 5 min the column is stripped with 100% acetonitrile and then regenerated with water. Eluant is monitored at 280 nm (range 2.0). Sample volume is 2 ml, and flow rate is 1 ml min - I .

Results

Vitelline protein B was purified by a combination of preferential extraction, ammonium sulfate precipitation, and reversed phase HPLC (Fig. 1). It is noteworthy that vpB consistently co-precipitates with approximately equimolar amounts of vpC during dialysis of the 20% ammonium sulfate-soluble fraction. Since the amounts of vpB in crude acid extracts of F. hepatica normally exceed vpC by a factor of about 10 [4], the co-precipitation step is either preferentially selecting a subgroup of vpB with high binding to vpC, or yields limited vpB by virtue of a saturation of vpB binding sites on vpC. We have yet to characterize the vpB not precipitated during dialysis. In any case, the 2 proteins precipitated were fully separable by HPLC using an acetonitrile gradient and a C-8 Aquapore column. Note that the peak corresponding to vpB is not symmetric, but rather is characterized by a steep ascent on the leading edge and a stepped descent on the trailing edge. Fifteen 1ml fractions (i-xv) under the peak were collected for analysis. Various kinds of polyacrylamide gel electrophoresis (PAGE) were performed to assess homogeneity in the peak

146

acid-urea

A

+

tris glycine-SDS

C

stacker -.9 97

kD

'"66 -... 43

1

~21

-I" . 14

i

B

iii

v

vii

ix

xi

xiii

i

iii

vii

ix

xi xiii

MW

isoelectric focus

D

tris borate SDS

v

+

-

, -"' 9 7 k D ,J=~,,~ -'.. 66 ~43

OIBII:BB

.......... ,,

1

-,1

{

--- 1 4

+ i

iii

v

vii

ix

xi

xiii

std

MW

i

iii

v

vii

ix

xi

xiii

pH

Fig. 2. Electrophoresisof vpB HPLC fractions (i to xiii, odd only) obtained from reversedphase HPLC. (A) Aceticacid/urea PAGE, about 5 fig protein per lane. Stain is CoomassieBlue R-250 in all panels. (B) SDS-PAGEusing Tris-borate, (C) SDSPAGE using Tris-glycine.(D) IsoelectricfocusingPAGE in the pH range 6~9. fractions. Acid-urea gel electrophoresis, which separates proteins by their size and charge density, reveals 2 sharp bands in all fractions (Fig. 2A). The upper band prevails throughout. P A G E in the presence of SDS and Trisglycine, which separates proteins according to their molecular weight, also resolves 2 proteins, this time with the lower band prevailing (Fig. 2C). Apparent sizes are 30 and 31.5 kDa. A smear of proteins extending from 31 kDa to 43 kDa is evident on SDS-PAGE in Tris-borate (Fig. 2B). This result probably reflects the strong binding of DOPA residues by borate and could conceivably modify the mobility of DOPA proteins in one of two ways: (1)

repulsion of SDS by the anionic catecholatoborate groups, and/or (2) increasing the apparent molecular weight by the number of complexed borate groups. If tyrosyl groups in the protein were completely hydroxylated to DOPA and the latter were all bound to borate, one might expect an increase in size of 500 Da. Thus, fractions having a high efficiency of tyrosine-to-DOPA conversion would appear to have a slightly higher molecular weight than those with low conversion. The amino acid compositions of nine fractions under the HPLC vpB peak do indeed reflect the prominence of DOPA in the earlier fractions, then gradually give way to tyrosine in the later

147 TABLE I The amino acid composition of vpB HPLC fractions with absorbance at 280 nm Amino acid Residues (per 1000) i

ii

iii

iv

v

vi

vii

viii

Asx Thr Ser Glx Pro Gly Ala Cys/2 Val Met Ile Leu DOPA Tyr Phe His Lys Trp Arg

140 18 50 85 17 177 70 0 5 23 3 40 120 13 32 41 113 0 53

153 17 50 87 17 170 70 0 5 22 2 39 112 19 30 40 115 0 52

150 17 51 87 19 183 70 0 5 22 3 40 93 28 29 39 112 0 51

150 17 51 88 17 171 69 > 1 5 22 3 39 84 42 37 39 114 0 52

151 18 52 88 20 176 70 0 6 22 4 40 72 45 29 40 112 0 53

152 18 52 88 18 175 70 0 6 22 3 40 73 47 28 39 117 0 52

150 17 50 86 19 188 69 0 5 21 3 39 68 50 29 40 112 0 54

153 17 52 87 19 174 69 0 5 22 3 39 74 47 30 41 114 0 53

152 18 51 87 18 177 70 0 5 23 3 40 67 52 31 40 113 0 52

Total

1000

1000

999

999

1000

1000

1000

999

999

T A B L E II The N-terminus of vpB fractions i~x (odd numbers only) sequenced through 28 cycles Cycle

l 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Amino acid

Arg His Pro His Gly Lys Phe Asn Arg His Ala Ser b DOPA/Tyr Asp Asp Arg Glu Lys His Arg Gly DOPA/Tyr Arg Lys Glu Asn Asp DOPA/Tyr

HPLC fraction i Yield a

iii Yield

v Yield

vii Yield

2.8 3.4 1.8 2.9 1.6 2.6 3.0 2.4 2.5 2.1 2.4 . 1.7/0.5 1.5 2. i 1.3 !.3 1.1 0.9 1.0 0.9 0.9/0.1 0.9 0.7 0.7 0.5 0.6 0.6/0.1

2.4 3.0 2.4 2.7 1.5 1.5 2.4 2.1 2.1 2.1 2.1 . 1.2/0.9 1.5 1.8 1.5 1.5 0.9 0.9 1.2 1.0 0.6/0.3 1.0 0.8 0.7 0.6 0.5 0.4/0.1

2.0 2.3 1.7 2.0 1.0 1.4 1.6 1.4 1.5 1.3

1.4 1.8 1.6 1.6 1.2 1.2 2.0 1.6 1.6 1.4

1.5

1.5

1.2 1.2 0.9 1.1 0.5 0.7 0.9 0.8 0.9 0.8 0.8

0.7/0.7 1.0 1.4 0.9 0.9 0.6 0.6 0.7 0.7 0.5/0.3 0.7 0.5 0.5 0.5 0.4 0.4/0.3

0.6/0.9 1.2 1.6 1.0 1.0 0.6 0.6 0.8 0.8 0.2/0,2 0.7 0.5 0.6 0.4 0.5 0.2/0.2

0.3/0.5 0.6 0.6 0.6 0.5 0.4 0.3 0.4 0.4 0.2/0.2 0.3 0.3 0.4 0.3 0.2 < 0.1/0.2

.

aYield in nmol. bSer is largely destroyed by the Edman degradation.

ix Yield

.

ix

148

C-8A

/ /t

o

E t

0

cD

34

7

~ 9

o

t5

l

13

10

30 45 Elution time (rain}

II

60

75

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

G-G-G-Y*-G-G-Y*-G-K G-G-G-Y*-D-S-Y*-G-K Y*-S-Y* E-T-P-Y*-A-R S-Y*-D-D-Y*-D-T-K Y*-E-D-D-Y*-A-R F-D-S-Y*-G-K Y*-E-P-Y*-G-R Y*-E-E-A-G-K Y-D-M-Y-G-E-R E-T-P-Y-D-K A-Y*-L-H-G-S-F-D-K K-E-N-D-Y*-L-N-Y*-D-L-K E-N-D-Y*-L-N-Y*-D-L-K Y*-M-F-D-S-Y*-G-K F-D- M-Y*-G-N-V-K A-D-G-Q-A-I-S-N-G-N-M-N-A-Y*-G-M-F-D-S-Y*-G-K

11.

90

Fig. 3. Separation of tryptic vpB peptides by C-8 reversed phase HPLC. Acetonitrile begins at 0%, increases to 4% at 9 min, 15% at 70 min and finally 31% at 75 min. After 75 min the column is stripped with 100% acetonitrile for 5 min and regenerated with water. Eluant was monitored at 280 nm (range 0.2). Sample volume was 2.0 ml and flow rate was 1.0 ml m i n - . The bracketed fractions (peak A) were pooled, lyophilized and rerun on a C-18 column.

ones (Table I). It is isoelectric focusing under denaturing conditions that resolves the highest level of heterogeneity. Twenty or more distinct bands are evident and stain for protein as well as D O P A (Fig. 2D). These have apparent pIs ranging from 6.9 to 8.3, respectively. The fact that these pIs are significantly broader than the pI of 7.4 reported earlier for vpB [6] is probably due to a more effective denaturation of the proteins by 8 M urea and 1% Triton X100 used in the present studies. The mass of

12. 13. 14. 15. 16. 17.

Fig. 5. Sequence of tryptic peptides of vpB resolved by reversed phase chromatography on C-8. Standard single letter code for amino acids is as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; Y, Tyr. Y* denotes DOPA.

2. 1.

Y-E-K G-Y-R Y-D-D-Q-G-K Y*-D-Q-Y*-G-K G-Y*-G-G-S-S-A-A-S-K F-A-N-K G-Y*-M-K A-Y-G-N-E-D-E-G-A-K

3. 4. 5. 6. 7. 8.

9. H-A-S-Y-D-D-R

10.

S-E-N-Y*-G-N-A-R

Fig. 6. Sequence of tryptic peptides of vpB resolved by reversed phase HPLC on C-18.

s

C-18

s S

s

s

s

o;

t

C-8B

s S sj

E c o o3 oJ c o

,.Q

S

12 ~

s S

oF

, ~ 1"10

,,;

O

?

~S1SSSSs

I

20

40 50 6 0 30 Elution time (min)

~>

o I

I I

< i

I0

I~ l

.Ibl

0 0

13~l

70

80

Fig. 4. Separation of tryptic vpB peptides not binding to C-8 by C-18 reversed phase HPLC. Acetonitrile begins at 0% and increases to 25% at 60 min; stripping with 100% acetonitrile ensues between 63 and 65 min returning to 0% at 68 min. Sample volume was 0.2 ml and flow rate was 1.0 ml.

,

IC) ' 20

i

i

i

i

i

,

i

30 4 0 50 60 Elution time (min)

i

L

70

i

80

90

Fig. 7. Separation of tryptic vpB peptides produced by digestion in the presence of 0.15 M borate. Acetonitrile begins at 0%, increases to 4% at 9 min, 15% at 70 min and finally 31% at 75 min. After 75 min the column is stripped with 100% acetonitrile for 5 min and regenerated with water. Eluant was monitored at 280 nm (range 0.2 t. Sample volume was 0.2 ml and flow rate was 1.0 ml min .

149

T A B L E III Peptide sequences obtained in the present study c o m p a r e d to those derived f r o m c D N A sequences o f putative eggshell precursors [ 1 4 , 1 6 l 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

G-Y*-G-G-S-S-A-A-S-K S-E-N-Y*-G-N-A-R G-H-Y*-S-L-A-G-K G-K-Y*-D-A-Y*-G-K S-Y*-D-D-Y*-D-T-K S-A- H-D-G-K°Y*-D-M-Y*-G-(R) F-D-M-Y*-G-N-V-K E-G-T-K-F-E-E-Y*-T-K F-D-S-Y*-G-K-Y*-D-Q-Y*-G- K E-T-P-Y-D-K-Y*-S-Y* A-Y*.L-H-G-S-F-D-K K-E-N-D-Y*-L-N-Y*-D-L-K S-K-F-D-L-Y*-G-N-V-E-A-K

vpB

Tryptic peptides

M a t c h i n g c D N A sequences vpB-1

C-8A

vpB-2

1 2 3

+ +

+ +

4

-

-

5

+ + + +

+ + + + +

-

+ + + + + +

6 7 8 9

Zurita et al. ( 1 9 8 7 ) +

+

F i g . 8. Peptide sequences o f vpB digested with trypsin in 0 . 1 5 M borate. A m i n o acids in parentheses denote residues

l0 11

detected by a m i n o acid analysis but n o t sequencer.

12 13 14 15 16 17

+ + + + + +

1 2 3

+ + +

4

+

+

5 6 7

+ + -

+ + +

+

8 9

+

-

+ +

+ +

+ +

protein centered around pH 8.0 in the earlier fractions appears to be precipitating in the gel. The tyrosine-to-DOPA modification is unlikely to contribute much to the isoelectric points of the proteins since the pKas of the phenolic OH groups of tyrosine and DOPA are well above the observed protein pls. In order to examine the basis for heterogeneity, we determined the amino acid sequence for 28 cycles into the N-terminus of each of the fractions i to xii under the HPLC peak of Fig. 1. As shown in Table II, with the exception of the complementary relationship of Tyr/DOPA (at cycles 13, 22, and 28), all fractions revealed the same sequence up to residue 28. D O P A prevails in the lower numbered fractions, whilst Tyr tends to prevail in the higher numbered fractions. This suggests that all variants collectively have identical amino termini prior to post-translational modifications. The second aim of this research was to explore peptidyl-DOPA sequences by improving the digestibility of vpB with trypsin. Earlier attempts at digestion were carried out in 0.1 M Tris HC1, pH 7.8 [6]. Despite the high proportion of Arg and Lys in the protein, only a handful of DOPA-containing peptides were recovered after 24 h of digestion, the bulk of the protein having remained intact. All the peptides produced under these conditions were

C-18

10 C-8B

1 2 3

--

--

--

+

8 9 10 11 12 13

+ + +

+ +

see C-18, 5 s e e C - 1 8 , 10

4 5 6 7

[+]

[xl

see C-8A, 5 + s e e C - 8 A , 16

[+] + see C-8A, + +

+ + + 12 +

--

+ +

[ + ] D e n o t e s sequence m a t c h e s that are n o t tryptic peptides, whereas [x] are peptides with imperfect sequence matches.

Gly-rich [6]. In order to circumvent the insolubility of protein and to bring about a more complete digestion of vpB, borate buffer was used in place of Tris. The digestion by trypsin was repeated, this time in 0.05~). 15 M borate pH 8.5 with 4 M urea. At borate

150

concentrations of 0.05 M, trypsin digests the protein extensively as shown by the number of peptides in Figs. 3 and 4. These peptides were first resolved by HPLC on a C-8 column (Fig. 3). Those peptides not binding the C-8 (peak A in Fig. 3), were further resolved on a C-18 column (Fig. 4). DOPA-containing peptide sequences are shown in Figs. 5 and 6. In contrast to earlier proposals, DOPA occurs in a variety of sequences, not just those flanked by glycine. On the amino side of DOPA, Ser and Lys occur with moderate frequency (20% each) as does Asp (14%), whereas on the carboxy side, Gly prevails (40%), followed by Asp (25%). Additional trypsin digests were performed in 0.15 M borate to determine if raising the borate concentration would further improve digestibility of vpB; peptide elution profiles and sequences are shown in Figs. 7 and 8, respectively. Note that the higher borate concentration has the effect of inhibiting cleavage of the sequence -Lys/Arg-Dopa-.

Discussion

Vitelline protein B consists of a closely related family of proteins that owes its apparent heterogeneity to variations in the Tyr-to-DOPA conversion as well as to subtle variations in the primary sequence. All variants fall within a narrow molecular weight range (30-33 kDa), a pI range of 6.9 to 8.3, and the majority would appear to collectively share a common N-terminal sequence up to 28 residues long. This N-terminus is identical to the sequence of putative eggshell precursors from F. hepatica as predicted from 3 c D N A sequences [14,15] provided that cleavage of signal peptides occurs at Arg 18 of the protein sequence based on the c D N A [16]. The Nterminal sequence of the 3 eggshell precursor transcripts, however, diverges after residue 51 (Lys) or 31 sans signal peptide. The DOPA/ Tyr-containing tryptic peptides described in this study fall into one of four categories vis-fivis the derived sequence of the three putative eggshell precursors (see Table III). (A) Those

with sequences common to all three. This is ostensibly limited to SY*DDY*DTK, FANK, H A S Y D D R , GYR, and K E N D Y L N Y D L K (6 of 36), although peptides such as G G G Y * G G Y * G K and G G G Y * D S Y * G K come very close. (B) Those with sequences that match only 2 of the 3 transcripts (17 of 36); (C) those with equivalent sequences in only one transcript (7 of 36), and (D) those with sequences matching none of the cDNAderived sequences (4 of 36). The origin of vpB heterogeneity is not at all clear at present. Allelic differences are discounted since individuals display the same heterogeneity as batch preparations [4]. Multiple gene copies, genomic rearrangement or post-transcriptional splicing are all possibilities, in principle. Using the c D N A of one putative eggshell precursor as a hybridization probe for screening a genomic library, Bobek et al. [15] found evidence for multiple, i.e., 5, but not verbatim, copies in female blood flukes Schistosoma mansoni. As many as 7 copies of VpB eggshell protein genes sharing a common N-terminus may be present in the genomic D N A of F. hepatica [16]. Additional variants of these genes may arise by alternative R N A splicing such as that described for silkworm sericins [17], type XIII collagen [18], and Drosophila eggshell proteins [19]. LoVerde et al. [20] suggest that the presence of multiple gene copies may parallel gene amplification in Drosophila, for example, in serving to express copious amounts of protein in response to developmental cues. Other functions for the observed microheterogeneity, however, such as a means of escaping destruction by the immune system or as fulfilling subtle morphological roles in the formation of eggshells, can not be discounted. The meaning of the variation in the Tyr-toDOPA conversion is difficult to assess at present. A similar variation has been observed in the post-translational modifications of an adhesive protein of the ribbed mussel Geukensia demissa [21]. Taken together, isoelectric focusing data and (DOPA/Tyr) ratios for the individual fractions under the HPLC peak suggest that all of the variants are indiscriminately modified. Perhaps the hydroxylating

151

enzyme in this system has a rather broad substrate specificity, but catalyzes Tyr hydroxylation in some sequences more rapidly than in others. Our results suggest that DOPA is more likely to occur when flanked by Lys, Ser, or Asp on the amino side and by Gly or Asp on the carboxy side. In contrast to other DOPA-containing proteins which have highly conserved peptide repeats [3], vitelline protein B peptides have an unmistakably degenerative primary sequence. The most frequently observed motif is (X1)-Tyr*-(X2)-Tyr*-(X3)-Lys/Arg where X1 represents 0-3 amino acids, X2 is usually 2-3 amino acids at least one of which is Asp or Gly, and X3 is one or two amino acids particularly Gly, Asp or Ala. Tyr* denotes that tyrosine can occur as the unmodified amino acid or as DOPA. This sequence resembles previously characterized DOPA-containing peptides from other organisms in that it ends in a basic amino acid (usually lysine) and has 2 DOPA residues separated by 2-3 amino acids per repeat [3]. It differs particularly in having 'spacer' amino acids between the second DOPA and C-terminal Lys (or Arg).

Acknowledgements The Office of Naval Research and the National Institutes of Health (Tropical Medicine and Parasitology and Restorative Materials) provided financial support for this research. Drs. Kathleen Little and Will Eppes generously assisted with liver fluke dissections.

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