Neuropeptides (1992) 21, 105-l 18 @ Longman Group UK Ltd 1992

Chromogranin A Epitopes: Clues from Synthetic Peptides and Peptide Mapping B. M. GILL, J. A. BARBOSA, R. HOGUE-ANGELETTI,

N. VARKI and D. T. O’CONNOR

Departments of Medicine and Pathology and Center for Molecular Veterans Affairs Medical Center, San Diego, CA, USA. Department Cancer, Albert Einstein College of Medicine, Bronx, NY, USA

Genetics, University of California, of Developmental Biology and

and

Abstract-Chromogranin A (CgA) is a 4BkDa acidic protein in neuroendocrine secretory vesicles whose primary structure is now known. We used synthetic peptides, synthetic peptide antisera, intact molecule antisera, chymotryptic peptide mapping, microsequencing, immunoblotting, and immunoprecipitation to probe the location of immunodominant domains within the CgA molecule. Polyclonal anti mid-molecule, anti N-terminal and anti C-terminal antibodies specifically visualized CgA (both bovine and human) in one and two dimensional immunoblots of adrenal chromaffin vesicles, and the stain CgA fragments further suggested bidirectional (both N- and C-terminal) cleavage or processing of CgA. Anti intact CgA immunoblotting of HPLC-separated peptides from chymotrypsin-digested bovine CgA revealed several strongly immunoreactive internal peptides, two of which were positioned by N-terminal amino acid sequencing: CgAs,n. and CgA,s,ti.. A single synthetic peptide (CgA79-113) was recognized by three antibodies developed against the intact CgA molecule: two polyclonal rabbit antisera as well as a monoclonal mouse antibody. Notlall antigenicity algorithm-predicted domains were immunogenic, suggesting that some of these predicted domains may not be accessible. Polyclonal anti mid-molecule, anti N- and anti C-terminal synthetic peptide antisera specifically immunoprecipitated ‘251-labeled bovine CgA from aqueous solution; mid-molecule antisera precipitated substantially greater amounts than terminal antisera. The immunoprecipitation results suggested exposed terminal as well as interior hydrophilic epitopes in the molecule in its intact, native conformation. ‘251-human CgA was best precipitated by anti N-terminal antisera, consistent with greatest interspecies sequence conservation at the N-terminus of CgA. The terminal antisera reacted immunohistochemically in a granular pattern with adrenal medullary chromaffin cells (but not adrenal cortical cells) and pancreatic islet cells (but not pancreatic exocrine acini). Thus, synthetic and chymotryptic peptides yielded novel and specific insights into the structure, conformation, vesicular processing and interior immunodominant domains of CgA. Date received 24 October 1991 Date accepted 6 November 1991 Supported by the Department of Veterans Affairs and the National Institutes of Health (HL-35018 and NS-22697).

Address for correspondence: Daniel T. O’Connor, M.D., Department of Medicine (V-111-H) University of California, San Diego, 3350 La Jolla Village Drive, San Diego, CA 92161, USA

105

106 Introduction

Chromogranin A (CgA) is a 48 kDa (1,2) acidic protein found in the core of and released by exocytosis from virtually all secretory vesicles in the neuroendocrine system (3-8). CgA’s full length primary structure has recently been deduced from its cDNA sequences in several species (1,2,9-14). The distribution and vesicular processing of CgA have been characterized in several tissues and species with anti intact molecule CgA antisera (15-17). However, it remains to be seen which region(s) of CgA the anti intact molecule antisera recognize; thus, such antisera may be of limited value in discriminating species- or tissue-specific patterns in CgA occurrence or processing. By generating antisera against synthetic fragments of CgA, and by examining CgA peptides (both proteolytic and synthetic) for reactivity against intact antisera, we have defined immunodominant domains within CgA, and have used CgA’s epitopes to examine the conformation and distribution of CgA, as well as processing of CgA within its storage vesicles.

Methods Proteins

Chromaffin vesicle soluble core lysates were prepared from normal bovine adrenal medulla, normal porcine adrenal medulla, and human pheochromocytoma as previously described (18, 19). Transmission electron microscopy of hormone storage vesicle fractions was performed as previously described (19). Bovine and human CgA were isolated from chromaffin vesicle soluble lysates as previously described (19,20), and rabbit polyclonal antisera directed against bovine and human CgA were produced as previously described (6, 20, 21). Chromogranin terminology was according to the guidelines previously outlined (22). Peptides and sequence analysis

The bovine CgA sequence was analyzed by a variety of algorithms to predict antigenic or immunogenic domains, using the IBI-Tustell

NEUROPEPTIDES

sequence analysis programs (International Biotechnologies Incorporated, New Haven, CT) on an IBM-PC-AT microcomputer. The algorithms included the antigenic index (23), flexibility (24)) hydropathy (25)) surface probability (26), and secondary structure by the formulas of Chou and Fasman (27) and Garnier et al (28). The antigenic index (23) is an expression which incorporates contributions from hydrophilicity, surface probability, backbone flexibility, and secondary structure predictions. The CgA sequence data used for these predictions included the N-terminal amino acid sequence of bovine and human CgA (29), the N-terminal sequence of rat pancreatic islet cell beta-granin (30), the amino acid sequence of porcine pancreastatin (31) and the cDNA-derived sequence data of bovine, porcine, rat and human CgA (1, 2, 9, 14). Peptides were synthesized on solid-phase supports from Boc- or Fmoc-protected amino acids (32), cleaved by HF from the resin, extracted, lyophilized, resuspended in 0.1% trifluoroacetic acid (TFA), and purified by reverse-phase high pressure liquid chromatography (RP-HPLC) on a semi-preparative 1 X 25cm C-18 silica column (Supelco), using a linear (O-60%) acetonitrile (CH&N) gradient in 0.1% TFA, monitoring absorbance at 280nm (Azso). Purity of the peptide eluants was verified by re-injection of the main A2s0 peak onto the same column, confirming the presence of only one peak on the second pass. Peptides were quantitated gravimetrically in tared flasks after lyophilization. A single adventitious terminal tyrosine was included in several peptides as a carrier-coupling moiety (see below), also enabling detection by A2s0 as well as labeling by radioiodination. A 14C-labeled residue (usually glycine) was single also included in most peptides (final specific activity, approximately lOOOOdpm/mg peptide), to quantitate efficiency of incorporation (coupling) of peptide hapten into the carrier (keyhole limpet hemocyanin) prior to immunization. Peptide structure was verified by analysis of amino acid composition of 24 h HCl hydrolysates (6, 19). All peptides’ measured amino acid compositions (residues/mole) agreed with predicted compositions (data not shown).

CHROMOGRANIN

EPITOPES

Polyclonal rabbit antisera

Peptide haptens were coupled to the carrier keyhole limpet hemocyanin (KLH; Calibiochem, La Jolla, CA), with percent coupling efficiency estimated by incorporation of non-dialyzable 14C-glytine into the carrier. The coupling agents were bisdiazobenzidine (BDB; Sigma, St. Louis, MO; for tyr-tyr linkage), 1(3-dimethylamino-propyl)-3ethylcarbodiimide (EDAC; Sigma; for amide linkage), or glutaraldehyde (Sigma; for amino linkage) (33). Typical peptide incorporations were: 41% for glutaraldehyde coupling, 26% for BDB coupling, and 14% for EDAC coupling. Male New Zealand white rabbits were immunized in multiple intradermal sites (34) at one month intervals with lmg of hapten coupled to carrier. The immunogens were emulsified in an equal volume of either complete Freund’s adjuvant (initial injection) or incomplete Freund’s adjuvant (subsequent booster injections). Antisera were harvested two weeks after each immunization, by central ear artery puncture. Antisera to the chromogranin A intact molecule (bovine or human) were developed by intradermal immunization of New Zealand white rabbits, as previously described (6, 20, 21). Monoclonal

mouse antibodies

Murine monoclonal antibodies were raised against the intact molecule bovine chromogranin A by established procedures (35, 36). Antibodies were obtained as ascites fluid (antibody 6F2) after peritoneal propagation of a hybridoma. Immunoprecipitation

Bovine and human CgA were radioiodinated with Na i2’l, using the chloramine T method (37), and isolated by gel filtration on Sephadex G-50 (38). Immunoprecipitation of ““I-CgA by rabbit antisera (directed against CgA or its synthetic fragments) was accomplished after an overnight incubation in 600~1 at 4”C, using an excess titre of second antibody (goat anti rabbit gamma globulin; Scantibodies, Lakeside, CA) and 1.7% (w/v, final concentration) polyethylene glycol, as described (3X). Immunoblotting

The ability of the peptide antisera to recognize

107 CgA size forms was evaluated by immunoblotting, as previously described (39). After SDS-PAGE of purified CgA or hormone storage vesicle soluble core lysates, proteins were electrophoretically transferred to nitrocellulose sheets, then immunoblotted and visualized via an avidin-biotin-horseradish peroxidase complex (Vectastain, Vector Laboratories, Burlingame, CA). Total proteins were stained by treating unblocked nitrodellulose sheets with amido black (Sigma) (39). Two dimensional gel electrophoresis was performed as described (40). The reactivity of both polyclonal rabbit antisera and monoclonal mouse ‘antisera against bovine CgA was confirmed by immunoblotting after one-dimensional SDS-PAGE (39). Peptide mapping of bovine CgA epitope dbmains

Gel exclusion-purified (20) bovine CgA (12Oug. 2.4nmol) was digested overnight with 5ug of chymotrypsin (Type VII, TLCK treated, 49 units/mg; Sigma), at an enzyme to substrate mass ratio of 1:24 (w/w). The digestion was performed at 25°C in 1OOmM CaCl2,80mM Tris, pH 7.8 (41). After adding 5 mM dithiothreitol (final concentration) the reaction was terminated by heating to 100°C for Smin. The heat treated sample was centrifuged in a microfuge (13000g) for 5 min at 25°C. One-half of the resulting CgA chymotryptic peptides (60ug; 12nmol) was subjected to RPHPLC on an analytical 250 x 4.6mm RP-304 (HiPore, BioRad, Richmond, CA) C-4 column, using a linear acetonitrile gradient in 0.1% TFA. Peak absorbance was measured at 214nms(AZi4). Collected fractions were lyophilized to dryness and each was resuspended in 10~1 of 0.1% TFA. One-third (approximately 400pmol) of each RP-HPLC fraction was pipette-spotted onto nitrocellulose sheets and air dried. Other samples spotted and dried on the nitrocellulose sheets were 0.1 nmol each of these synthetic peptides (see Table 1) in 0.1% TFA: bovine/human CgAl.rh[tyri,], bovine CgAr.40, bovine CgA7v.r i3. porcine CgAz4o.&pancreastatin), human Cg#zh8-3iy (pancreastatin), bovine CgA332.3h4, bovine CgA367.391, bovine CgA403-42s and [tyr&bovine CgAdi,-431. Control spots (each pipetted in 0.1% TFA) were 0.2nmol (1Oug) of purified~ intact bovine CgA (positive control), 20ug of bovine

Mid-molecule (pancreastatin)

Porcine

Human

Bovine

Bovine

Bovine

Bovine

I

8

9

10

11

12

(GE25)

(LL33)

(MM)

C-terminal

Mid-molecule (PL26) [near C-terminus]

Mid-molecule

Mid-molecule

Mid-molecular (pancreastatin)

Mid-molecule (pancreastatin)

6

Mid-molecule

Bovine

(HE35)

5

N-terminal

Mid-molecule

2

N-terminal

Bovine

Rat

1

4

Bovine/

Number

Region in the CgA molecule (trivial name)

of chromogranin

N-terminal (LE40)

Species sequence

peptides

3

Synthetic

Table

CgAl.6-[tyr17]

a

YELEKVAHQLEELRRG

[tyro]-bovine CgA417.431

b

PEDQELESLSAIEAELEKVAHQLEEL

b

GWRPNSREDSVEAGLPLQVRGYPEE

b

Bovine Cg&3.428

LGEEEEEEDPDRSMRLSFRARGYGFR GPGLQL

b b

a

c (Allen, Portland, OR) b

b

b (HogueAngeletti, Bronx, NY)

A (O’Connor San Diego, CA) a

Synthesized

Bovine CgAx7.391

Bovine CgA332m364

EEETAGAPQGLFRGY

Porcine CgAZ75.288-(tyr15]

GESRSEALAVDGAGKPGAEEAQDPEG KGEQEHSQQKEEEEEMAWPQGLFR G

GWPQAPAMDGAGKTGAEEAQPPEGK GAREHSRQEEEEETAGAPQGLFRG

HPLASLPSPKYPGDQAKED

HSSYEDELSEVLEKPNDQAEPKEVTE EVSSKDAAE

LPVNSPMNKGDTEVMKCIVEVISDTLS KPSMPVSKECFE

LPVNSPMTY

LPVNSPMNKGDTEVMKY

Sequence

Porcine CgA2*2X8

Bovine CgA163.181

bovine CgA79.113

Bovine CgAI-dO

Rat CgAl.s-[tyrg]

Bovine/human

Residues in parent CgA molecule

A (CgA)

by

12

12

172

9,lO

14

14

12

132

132

11-13

1,2,9,13,29

Reference

CHR~M~ORAIW

109

EPITOFJE~

chromaffin vesicle lysate protein (-1Oug bovine CgA; positive control), -0.15nmol (1Oug) of bovine serum albumin (negative control), and 2.5~1 of 0.1% TFA alone (negative control). The nitrocellulose sheets were immunoblotted (as described above) with two anti intact molecule bovine CgA antisera (from two separate rabbits, immunized with two independent bovine CgA preparations) each at two titers (1:200 and 1: 10000, v/v), and an anti intact CgA molecule murine monoclonal antibody (6F2, hybridoma ascites) at a 1:lOOO (v/v) titre. Those HPLC-separated chymotryptic peptides (-400pmol of each peptide) that reacted against both anti intact molecule bovine CgA antisera at high titre (1: 10000) were subjected to N-terminal microsequencing (29) on an Applied Biosystems coomassie blue

Stain

Preimmune serum

j-i* Ktefmlnus

34

5

Fig. (CVL)

1

ma&Ma

Biosystems,

Immunohistochemistry

Human autopsy tissues, obtained with 12h of death, were embedded in freezing compound (OCT, Miles Scientific), then flash-frozen and stored at -70 degrees C. Ten microns frozen sections were mounted on gelatin-coated slides. Endogenous peroxidase was blocked by overlaying the tissue sections with 0.03% hydrogen peroxide for 10min. Blocking solution (10% [v/v] goat serum in 1% [w/v] bovine serum albumjn/phosphate-buffered saline, pH 7.4) was overlayed on the tissue sections for 30min to inhibit nonspecific antibody binding. Antisera at dilutions of g: 100 to 1: 1000 (v/v) were incubated with tissue section antigens for 1 h at room temperature. Unbound antibody was washed off with PBS. A peroxidaselabeled goat anti rabbit second antibody (Biorad Laboratories) was diluted in blocking solution and incubated with tissue sections for an additional hour. Unbound antibody was washed off, land the color reaction was developed using 3’-9’Caminoethyl-carbazole in 0.03% hydrogen peroxide. After a brief counterstain with hematoxylin, the sections were mounted using glycerol-gelatin (Sigma) and coverslipped for viewing under an Olympus BH2 microscope.

6

+

+

+

Immunoblotting

+ + -

+++ -

+

1: Size stsndsrds cdenal

(Applied

Results

Immunogen: 17-mar Smer Coupler: Glut. anE ProMsorption:

2-7 : Bmins

Model 670A sequenator Foster City, CA).

CVL

Immunoblotting of bovine chromaffin vesicle lysate soluble core proteins with antisera directed against CgA

synthetic peptides. The effect of peptide human CgA N-terminal l7-mer [Peptide N-terminal 9-mer [Peptide 2, Table]),

immunogen (bovine/ 1, Table] or rat CgA mode of coupling the

hapten to the keyhole limpet hemocyanin carrier (glutaraldehyde [glut] or bisdiazobenzide [BDB]), preadsorption of the antiserum with the 17-mer hapten (lOug/ml, 4°C overnight), and normal rabbit serum (preimmune serum) on bovine CgA immunoblotting are shown. CVL lanes contained 25ug of bovine CVL proteins. Each antiserum dilution was 1:lOO (v/v).

Anti N-terminal CgA antisera specifically recognized bovine CgA on chromaffin granule immunoblots, whether the immunogen was bovinel/human CgAt-r6-[tyr17] or rat CgAr-a-[tyrg], and regardless of the coupling procedure (Fig. 1). The specificity of staining is shown by the last 2 control lanes. The antibodies also recognized material of high r SDSPAGE M, that CgA, consistent with the C A-core proteoglycan (42,43), as well as lower SDS,i PAGE M, forms of chromogranin A immunoreactivity (vide infra). With serial dilution of the N-terminal or C-terminal antisera, all of the stained CgA size form bands faded in parallel, reinforcing their immunologic similarity. Preadsorption of individual

110

NEUROPEPTIDES Ani& black

Stain

Anti Kterninus

Anti CternitWS

Anti whde

molde

NRS & -CgA

b9s

”

27-

17-

Sanple

s v

preadscrpticn

-

svsv -

sv N

sv

C

svsv N

sv C

S=Standards V=Chromaffln N=N-terminel C=C-terminal

sv

sv

s

N

C

vesicle peptide peptide

lysate

v

Fig. 2 Specificity of immunoblot staining by antisera directed against synthetic CgA N- and C-terminal peptides. On the far left are shown amido black stains of molecular weight standards (S) or bovine adrenal CVL proteins. On the near left is staining by an antibody directed against bovine/human CgA N-terminal 17-mer (Peptide 1, Table) synthetic peptide (N), with or without preadsorption (4”C, 16h, 1Oug peptidelml) by N or by a bovine CgA C-terminal 16-mer (Peptide 1, Table) synthetic peptide (C). In the center is staining by an antibody directed against C, with or without preadsorption by N or C. On the near right is staining by an antibody directed against the intact bovine CgA molecule, with or without preadsorption by N or C. On the far right is staining by preimmune (normal rabbit) serum. The migration position of CgA is shown at the right.

antisera with the relevant peptide specifically abolished staining by the corresponding antibody alone. Terminal peptide preadsorption (Fig. 2) did not affect staining by the anti intact CgA antibody, nor did the anti intact CgA antibody recognize the terminal synthetic peptides after peptide adsorption to nitrocellulose sheets. Figure 2 also shows that a variety of lower molecular size forms of CgA immunoreactivity were stained by all 3 antibodies (anti N- and C-terminal, and anti intact molecule), suggesting bidirectional cleavage or processing of CgA in chromaffin granules. During immunoblots with two mid-molecule synthetic peptide antisera, an anti porcine pancreastatin fragment (porcine CgA27sV2ss-[tyr15]) antibody recognized CgA in pig but not human or bovine chromaffin vesicles, while anti bovine CgA163-181 stained bovine CgA as well as lower M, size forms, but not human CgA. Both N- and C-terminal CgA antibodies

detected immunoreactivity or larger apparent size (SDS-PAGE M,) on immunoblots than CgA itself. To evalute this form further, we separated chromaffin vesicle core proteins by two-dimensional gel electrophoresis (Fig. 3). The amido black protein stain (Fig. 3A) showed two other proteins of larger size than CgA: the M, lOOkDa, more alkaline chromogranin B, and a more acidic M, 80-90 kDa spot. Both the N-terminal antibody (Fig. 3B) and the C-terminal antibody (Fig. 3C) detected the acidic Mr 80-90 kDa spot as well as CgA. In other studies, this M, 80-90kDa form of CgA immunoreactivity has been identified as a CgA-core proteoglycan (42, 43). Peptide mapping of bovine CgA epitope domains

Chymotrypsin-digested bovine CgA was resolved by RP-HPLC into 15 fractions over a linear acetonitrile gradient (Fig. 4). When the RP-HPLC

CHROMOGRANIN

111

EPITOPES

7.5

IEF,

4.2 I

pH

7.5 I

4.2 I

IEF, pH

1307550-

2

-0 Y

\

s;

50-

& UJ 3g $ 27-

27-

ici fn 17-

17-

Sample: Stain:

C Sample: Stain:

A

bovine amido

CVL,

100

bovine CVL, 100 anti C-terminus #239-3, 1: loo

ug

ug

black

7.5

4.2

130752

50-

2& mx

3927-

17Fig. 3 lysates dimension isoelectric

Immunoblotting of bovine (CVL) after two dimensional

chromaffin vesicle gel electrophoresiq,

Isoluble where

I (horizontal) was charge separation by tube gel focusing, and dimension 2 (vertical) was sizeisepara-

tion by SDS slab gel electrophoresis, followed by electrhphoretic transfer of proteins onto nitrocellulose sheets. The $taining was by amido black (panel A), anti bovine/humah CgA

Sample: Stain: 6

bovine CVL, 100 anti N-terminus #784-2, 1: 100

ug

fractions were immobilized on nitrocellulose and immunoblotted with two different rabbit polyclonal anti intact molecule bovine CgA antisera, peaks 10, 11, 12 and 15 (asterisked in Fig. 5) reacted with each antibody at high titre (1: 10000). HPLC peaks 8, 9, 13 and 14 also reacted, but

synthetic [tyri,]; lo-mer

N-terminal

17-mer

panel B), or anti peptide ((tyru]-bovine

peptide

(bovine/human

CigAi.i,-

bovine CgA synthetic C-terminal CgA417113r; panel C).

showed substantially weaker immunoreactivity even at a more concentrated 1:200 antibody titre. Among the synthetic CgA peptides tested in this same immunoblot experiment (see Methods and Table), only bovine CgA79-r13 reacted with both rabbit polyclonal antisera at high titre. Positive

112

NEUROPEPTIDES Sample: bovine chromogranin A (60 pg). chymotrypsin-digested Column: C-4 (RP-304. I-h-Pore). 250 x 4.6 mm 0.256

Buffers: A = 0.1% CFsCGOH B = 0.1% CF@0-l Elution: 1 mllmin

in Hz0 in CHsCN

-

L 10

I

I

I

I

I

I

I

15

20

25

30

35

40

45

1 50

I

-'O

55

Retention time, min. Fig. 4 Separation of chymotrypsin-generated bovine chromogranin A (CgA) peptides by reverse-phase high pressure liquid chromatography (RP-HPLC). Bovine CgA (120ug) was digested overnight with chymotrypsin (Type VII) using an enzyme to substrate mass ratio of 1:24, w/w. Half (6Oug, 1.2nmol) of the chymotrypsin-digested bovine CgA was subjected to RP-HPLC on a 250 x 4.6mm analytical C-4 column (HiPore RP-304, BioRad, Richmond, CA). The chymotryptic peptides were loaded isocratically in 0.1% trifluoroacetic acid (TFA) for 4min., and eluted using a linear acetonitrile gradient in 0.1% TFA. Peptide peaks were monitored by absorbance at 214nm wavelength (AZ14) with a Waters Model 441 specrophotometer. Collected fractions (15 numbered peaks in chromatogram) were lyophilized and resuspended in 0.1% TFA. Approximately 400pmol of each RP-HPLC fraction was immunoblotted using two different anti intact bovine CgA antisera. Four fractions (fractions 10,l I, 12 and 15; asterisk above peak numbers) which immunoreacted with both antisera at high titer (1: 10000 dilution, v/v) were submitted for N-terminal amino acid sequencing. Sequence data (one letter code) were obtained for three fractions (peaks 10, 11, 12) and are shown above the corresponding peak.

controls (bovine CgA; bovine chromaffin vesicle lysate) reacted with both antisera at both titres, while negative controls (bovine serum albumin; 0.1% TFA alone) did not react with either antiserum at either titre. A monoclonal murine anti bovine antibody (6F2, ascites fluid at 1:lOOO titer) recognized the synthetic peptide bovine CgA79-113; this antibody did not visualize chymotryptic column fractions or other CgA synthetic peptides. Highly immunoreactive column fractions 10, 11, 12 and 15 (Fig. 4) were sequenced. Fraction 10’s sequence (EKPNDQAEPKEV. . .) corresponded to bovine CgA residues 91 and following. The sequences of fraction 11 (LXAEQGRQTEREE. . .) and fraction 12 (SAEQGRQTERE. . .) correspond to bovine CgA residues 196 and following, and 197 and following; thus they overlapped and were offset by only one residue. Fraction 15 yielded no sequence data.

Immunoprecipitation

Anti N-terminal CgA antisera from several rabbits precipitated ‘*?-labeled intact bovine CgA. Both anti bovine CgAl-16-[tyr17] and anti rat CgAI-s[tyrg] antisera were effective, regardless of hapten coupler [BDB, EDAC, or glutaraldehyde). In general, maximal titres occurred by the third or fourth boost and bleed. The maximal titre for most such antisera in this system was 1:300 (v/v), while working (that is, 50% of maximal binding, or half maximal) titres ranged to beyond 1:lOOO. Antisera to the intact CgA molecule as an immunogen precipitated 3-9 fold more labeled CgA than the anti N-terminal antisera at optimal titres. C-terminal ([tyro]-bovine CgA417-431) antiserum titers for immunoprecipitation of intact bovine CgA also rose progressively after multiple immunizations, with maximal titres of 1:300 and working titres in excess of 1:3000. By comparison, anti intact molecule and anti mid-molecule

CHROMOGRANIN

113

EPITOPES

tion. The anti intact molecule antibody precipitated at least 10 fold more CgA than the anti terminal peptide sera at similar titers. The anti N-terminal antibody precipitated 2.5-3 fold more human CgA than similar titers of the other region specific antisera.

r

6000

/

Epitopes: prediction

1000 -

versus experiment

Figure 6 summarizes the results of peptidle mapping and synthetic peptide approaches to determination of immunodominant regions of bovine CgA. and compares the experimental results to antigenicity predictions based on several algorithms.

500 -

Immunohistochemistry OAnlrbody (I 3GQ r,rer, to Blank Whole

+ 1,.me,

C-termma,

,iGner

Anl!gen preadsorphon NOIW N-fermmai C-fermmal

Fig.

+ +

N-termna,

ecule) bovine

+

mO,ec”ie

5

+

+ +

(lOOug/ml) +

+

+ i

aqueous synthetic

+ +

of “51-bovine

precipitation of “‘I-bovine aqueous solution by antisera synthetic terminal fragments

+

+

+

solution peptides.

+

+ +

+

Immunoprecipitation

(4°C. overnight) peptide haptens

+ +

77.mer 76.mer

from CgA

+

CgA

+

(intact

mol-

by antisera directed against The specificity of immunmo-

CgA (intact whole molecule) from directed against bovine CgA or its is shown. Antibody preadsorption

controls with are also shown.

synthetic

N-

and

C-terminal

(bovine CgAle3.,8,) antisera precipitated 2-4 times as much CgA as similar titres of the anti terminal antibodies. The specificity of the interaction of the terminal antisera with the native CgA molecule is demonstrated in Figure 5. Neither the preimmune serum background nor immunoprecipitation by the anti intact CgA antibody was altered by preadsorption with the N- or C-terminal peptides. Immunoprecipitation by the anti N-terminal antibody was blocked by the N-terminal hapten, but not by the C-terminal hapten. Conversely, the anti C-terminal antibody was blocked by the C-terminal peptide, though not the N-terminal peptide. These antisera also interacted with intact, purified human pheochromocytoma CgA in solu-

NELJRO.-C

In frozen histologic sections of normal ‘human adrenal medulla, an anti N-terminal (rat CgA,.x[tyrs]; BDB-conjugated) CgA antibody vistialized all adrenal medullary chromaffin cells in a granular pattern, but not adrenal cortical cells (Rig. 7). Staining was also found in human pancreatic islets. but not exocrine acini. Preadsorption of the antibody with the synthetic N-terminal peptide hapten (5Oug/ul antiserum) abolished the staining. An anti C-terminal CgA antibody ([tyroj-Ibovine CgA417.431; glutaraldehyde-conjugated) s;howed an adrenal medullary staining pattern (Fig. 7) similar to that of the anti N-terminal antibody. Staining was also found in pancreatic islets, but not exocrine acini. Staining was also abolisqed by preadsorption with the synthetic C-terminal hapten (5Oug/ul antiserum). Similar immunohistochemical results were obtained whether antibody binding was visualized directly with a peroxidase-conjugated second antibody (see Methods), or with an intervening avidinbiotin complex bridge (Vectastain. Vector Laboratories, Burlingame, CA). Preimmune (normal) rabbit serum gave no staining under these conditions. These specific antisera (anti N-terminus, anti C-terminus) did not stain formalin-fixed. parafin-embedded human tissues (adrenal, but neuroendocrine tumors) under the conditions described here. Discussion Several

solution

immunoprecipitation

and

114

NEUROPEPTIDES buv CgA

Chymotryptic restdues 91 Ii 1.. Peptides --------------------------------------------------------HE35 Synthetic LE40 Peptides =,a -by” CgA,,113

A lmmunoreactive with anti-intact bovine CgA

bv @A residues 196 Ii

m...

GE25

Pancreastatin human Wmm

i

z

L

CgAj.,,

LL33

porcine CgA2~-2~ ----

U

B

PL26

bw CgAmw b Wm3.m 00 bov. CSAX.~M

bov CgAIII131

--

rat CgA, B

lmmunoprecipitation of intact bovine CgA by synthetic peptide antibodies

WI-16

gA,,,,,l

t

I

I!

1

30

60

90

I

I

I

f

I

I

I

I

I

I

I

120

150

180

210

240

270

300

330

360

390

420

Residue

C

Bovine

Number

Chromogranin

A

307

Antigenic

Index

00 -0 7 t +l 2

Flexibility

+I 0

Hydropathy

Surface

00

Probability

ChouFasman c Garnier, et al.

+05

I3 Sheet n Helix Turns 0 Sheet n Helix

E

Turns

a m .

.

.

(

Fig. 6 Schematic map of actual (panels A and B) versus algorithm-predicted (panel c) antigenic determinants within bovine chromogranin A. (A) Chymoptryptic (Fig. 4) or synthetic (Table I) CgA peptides immunoblotted with anti intact bovine CgA antisera. The peptides were scored by antibody titer required for immunodetection: antibody titer > 1:lOOOO (solid box); 1:200 < antibody titer < 1:lOOOO (shaded box); undetectable even at 1:200 antibody titer (open box). Map positions for the chymotryptic peptides were assigned corresponding to N-terminal amino acid sequence data obtained. (B) Immunoprecipitation of intact bovine CgA by synthetic peptide antisera, expressed as percent of total “‘I-CgA immunoprecipitated at an antibody titer of 1:300 (the usual maximal titer; see Fig. 1). Synthetic peptide antiserum efficacies were scored as immunoprecipitation of >30% (solid box), or from 8-30X (shaded box) of total counts. (C) Computer algorithms (IBUPutstell) predicting antigenic index (23), flexibility (24), hydropathy (25), surface probability amino acid sequence. The CgA sequence

(26). and two different depicted is the mature

secondary structure protein after signal

(27,28) peptide

profiles cleavage.

based

on the bovine

CgA

CWROMOGRANIN

EPITOPES

115

B

Fig. mag the C-k

7 Immunohistochemistry m&cation was 500 diameters bottom right. (A) Staining trminal

lo-mer

([tyr,,]-bovine

of CgA (antisera at 1:5OU dilution, v/v) in frozen sections of normal human adrenal gland The on the 5” x 7” print. The medullary chromaffin cells are the top left, while the cortical dells are at with an anti rat CgA N-terminal Y-mer (rat CgA , x-[tyrg,]). (B) Staining with an anti bovine ‘&!A CgAIr, 43,).

116 immunoblotting results suggest that the immunodominant epitopes of CgA are not located at either terminus: (a) anti terminal antisera had lower immunoprecipitation titers than anti intact molecule antisera; (b) anti terminal antisera precipitated less of the labelled intact molecule than anti intact molecule antisera, even at maximal effective titers; (c) anti intact molecule immunoprecipitations were not affected by preadsorption with the terminal peptides (Fig. 5); (d) anti intact molecule antisera did not recognize the nitrocellulose-immobilized terminal peptides (peptides 1,2, 3, 11 and 12; Table), nor was anti intact molecule antiserum recognition of immunoblotted CgA affected by preadsorption with the terminal peptides (Fig. 2); and (e) anti intact molecule antisera showed strongest immunoreactivity with midmolecule domains of chymotrypsin-digested bovine CgA (Fig. 4). The interspecies crossreactivity of the peptide antisera was consistent with known interspecies differences in the primary structure of CgA (1, 2, 9-14). The N-terminus of CgA is highly conserved (1, 2, 9-14); hence, anti bovine/human CgAl.rh[tyr17] and anti rat CgAi-s-[tyrY] sera recognized both bovine and human CgA, in solution as well as on nitrocellulose (Figure 1). The C-terminus of CgA is less conserved (1, 2, 9-14); thus anti [tyra]-bovine Cg&i7-4ar immunoprecipitated human CgA relatively poorly. The porcine pancreastatin region recognized by anti porcine CgA275-28s-[tyr15] is only 71% homologous to bovine CgA (14), and 77% homologous to human CgA. Anti porcine CgA275-2ss-[tyr15] recognized porcine CgA, but not human or bovine CgA. By contrast, Peterson et al. (44) found that antisera directed against synthetic fragments of bovine CgA may have rather broad interspecies crossreactivity, detecting CgA-like immunoreactivity even in trichocysts of the protozoan Paramecium. The pattern of lower moleculer size CgA immunoreactivity in chromaffin granule immunoblots (Figs 2 and 3) suggested bidirectional CgA cleavage or processing. Furthermore, the predominance of fragments with preserved C-terminal staining suggests that CgA cleavage proceeds principally from the N-terminus inward. The Cterminal CgA fragments were somewhat more

NEUKOPEPTIDES

acidic than the N-terminal fragments (Figure 3). Wohlfarter et al. (45) have also concluded from synthetic peptide antiserum immunoblots that CgA processing is bidirectional. In a separate report, we have isolated and characterized some of these CgA cleavage products (42). By immunoblotting chymotryptic CgA peptides (Fig. 4)) we identified at least two separate regions in the interior of CgA that may be immunodominant epitopes. The only synthetic peptide immunoreactive with several anti intact bovine CgA molecule antisera (both polyclonal rabbit and monoclonal murine) was bovine CgA,‘). , , .i. Because bovine CgA79-1r3 overlaps the sequence of cymotryptic peak 10 (Figs 4 and 6) at bovine CgA residues 91-102, we suggest that this short shared region may represent an immunodominant epitope. The immunodominant domain sequences obtained from chymotryptic CgA fragments (Fig. 4) are in regions of the CgA molecule that are ill-conserved across species lines - comparing bovine and rat CgA primary structures (13), the sequence EKPNDQAEPKEV . . . (Fraction 10) is only 17% (2/12 residues) conserved, while the sequence SAEQGRQTERE . . . (Fractions 11 and 12) is only 18% (2/11 residues) conserved. Thus, the reported variability in interspecies recognition of CgA by antisera directed against another species’ intact CgA immunogen (4,5,46) is predictable. In Figure 6 the correspondence of actual and algorithm-predicted antigenic (immunodominant) domains is depicted. In general, those domains experimentally determined to be most antigenic (based on either synthetic peptide or chymotryptic peptide immunoreactivity) were also areas predicted to be antigenic by the antigenic index (23), flexibility (24), hydropathy (25), or surface probability (26). Thus, antigenicity prediction algorithms (Fig. 6) would appear to be applicable to protein members of the chromogranin/secretogranin family (22). However, the Chou-Fasman (27) and Garneri (28) algorithms differed in secondary structure prediction of turns at one of the bovine CgA molecule’s most immunoreactive regions, as represented by residues 91 and following (peak 10, Fig. 4) and bovine CgA79.rr3. Furthermore, despite the extreme hydrophilicity of most of the span of the

CHROMOGRANIN

EPITOPES

117

tissues and in serum. Regul Peptides 6: 263-80. mature CgA protein, with a substantial number of 4. O’Connor, D. T., Burton, D. W. and Deftos, L. J. (1983). predicted surface domains (Fig. 6 [l, 13]), only a Chromogranin A: immunohistology reveals its universal very few regions of the molecule are consistently occurrence in normal polypeptide hormone producing highly immunogenic. This finding suggests that at endocrine glands. Life Sci. 33: 1657-63. least some of these predicted domains may not be 5. O’Connor, D. T., Burton, D. W. and Deftos, L. J. (1983). Immunoreactive chromogranin A in diverse olypeptide fully accessible to immunocompetent cells, in turn hormone producing human tumors and norm ifI endocrine implying a more complex secondary structure of tissues. J. Clin. Endo. Metabl;. 57: 1084-6. CgA than is usually ascribed to this molecule, 6. O’Connor, D. T. and Frigon, R. P. (1984). Chromogranin which has some of the attributes of a random coil in A, the major catecholamine storage vesicle soluble prosolution (6,47). Indeed, CgA secondary structure tein. Multiple size forms, subcellular storage, and regional distribution in chromaflin and nervous tissues elucidated prediction by the algorithms of Chou and Fasman by radioimmunoassay. J Biol Chem 259: 3237147. (27) and Garnier et al. (28) reveal substantial areas 7. Lloyd, R. V. and Wilson, B. S. (1983). Specific endocrine of organizaed alpha helical and beta pleated sheet tissue marker defined by a monoclonal antibody. Science structure (Fig. 6). 222: 628-30. Within the eight-exon CgA gene, the epitope 8. Cohn, D. V., Elting, J. J., Frick, M. and Eldes R. (1984). Selective localization of the parathyroid secretory protein beginning at amino acid residue 91 or spanning I/adrenal medulla chromogranin A protein family in a wide CgA79-ii3 (Fig. 4) is located towards the 3’ end of variety of endocrine cells of the rat. Endocrinology 114: exon 5, while the epitope beginning at amino acid 1963-74. residue 196 is located in mid exon 6 (48). Neither 9. Konecki, D. S., Benedum, U. M., Gerdes, ‘H. H. and of these highly immunoreactive epitopes is conHuttner, W. B. (1987). The primary structure of human chromogranin A and pancreastatin. J Biol Chem 262: gruent with CgA fragments of known biologic 17026-30. activity, such as pancreastatin (bovine CgAZ4seZg4 10. Helman, L. J., Ahn, T. G., Levine, M. A., Allison, A., [49]) or chromostatin (bovine CgA124-143 [50]). Cohen, P. S., Cooper, M. J., Cohn, D. V. and Israel, M. In conclusion, synthetic and chymotryptic pepA. (1988). Molecular clanging and primary structure of tides yielded specific insights into the structure, human chromogranin a (secretory protein I). JiBiol Chem 263: 11559-63. conformation, distribution, post-translational modifications and immunodominant epitopes of 11. Hutton, J. C., Nielsen, E. and Kastern, W. (1988). The molecular cloning of the chromogranin A-like precursor of CgA. beta-granin and pancreastatin from the endocrine pan-

Acknowledgements We appreciate the technical support of Siv Garrod, Michelle Deguire, Thai Dinh, James Pierce and Annie Chen. Dr. Russell Doolittle of UCSD synthesized peptides 1, 2, 7 and 9 (Table). Chymotryptic peptides were sequenced in the laboratory of Dr. Susan Taylor, UCSD. Dr. Richard Allen (Institute for Advanced Biomedical Research, Portland. OR) kindly supplied antiserum MM, directed against bovine CgAL62.1XI.

References 1. Benedum, U. M., Baeuerle, P. A., Konecki, D. S., Frank, R., Powell, I., Mallet, I. and Huttner, W. B. (1986). The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells. EMBO J 5: 14951502. 2. lacangelo, A., Affolter, H. U., Eiden, L. E., Herbert, E. and Grimes, M. (1986). Bovine chromogranin A sequence and distribution of its messenger RNA in endocrine tissues. Nature 323: 82-6. 3. O’Connor, D. T. (1983). Chromogranin: widespread immunoreactivity in polypeptide hormone producing

NEURO. -D

creas. Febs Letters 236: 269-74. 12. Iacangelo, A., Okayama, H. and Eiden, L. E. (1988). Primary structure of rat chromogranin A and distribution of its mRNA. Febs Lett 227 (2): 115-21. 13. Parmer, R. J., Koop, A. H., Handa, M. T. andG’Connor. D. T. (1989). Molecular cloning of chromogranin A from rat pheochromocytoma cells. Hypertension 141 435-444. 14. Lacangelo, A. L., Fischer-Colbrie. R., Keller, K. J., Brownstein, M. J. and Eiden. L. E. (1988). The sequence of porcine chromogranin A demonstrates chromogranin A can serve as the precursor for the biologidally active hormone, pancreastatin. Endocrinology 122: 2339-41. 15. Hagn, C., Klein, R. L., Fischer-Colbrie. R., Douglas. B. H. and Winkler, H. (1986). An immunological characterization of five common antigens of chromaffin gdanules and of large dense-cored vesicles of sympathetic nerve. Neurosci. Lett. 67: 2905-300. 16. Schober. M.. Fischer-Colbrie, R., Schmid, K. W.. Bussolati, G., O’Connor, D. T. and Winkler, H. (1987). Comparison of chromogranins A. B, and secretogranin II in human adrenal medulla and pheocrhomocy/toma. Lab Invest 57: 385-91. 17. Fischer-Colbrie, R. and Frischenschlager, 81. (1985). Immunological characterization of secretory proteins of chromaflin granules: chromogranins A. chromogranins B.

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small doses of immunogen. J Clin Endo 33: 988-91. 35. Goding, J. W. (1986). Monoclonal antibodies: principles and practice. 2nd edition. Academic Press, London, England. 36. Angeletti, R. H., Bilderback, M. and Qian, J. (1987) Antibodies to a synthetic peptide, chromogranin Ai.r.,. Ann NY Acad Sci 493: 138-40. 37. Odell, W. D. (1983). Radiolabelling techniques. Chapter 5, in: Principles of competitive protein binding assays. 2nd edition. John Wiley, NY, pp 69-84. 38. O’Connor, D. T., Pandian, M. R., Carlton, E., Cervenka, J. H. and Hsiao, R. J. (1989). Rapid radioimmunoassay of circulating human chromogranin A: in vitro stability, exploration of the neuroendocrine character of neoplasia, and assessment of the effects of organ failure. Clin Chem 35: 1631-7. 39. Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci, USA 76: 4350-4. 40. O’Farrell, P. A. (1975). High resolution two-dimensional gel electrophoresis of proteins. J Biol Chem 250: 4007-21 41. Worthington enzymes and related biochemicals. (1982). Biochemical Products Division, Worthington Diagnostic Systems Inc., Freeland, NJ, pp 44-5. 42. Barbosa, J. A., Gill, B. M., Takiyyuddin, M. A. and O’Connor, D. T. (1991). Chromogranin A: Posttranslational modifications in secretory granules. Endocrinology 128: 174-190. 43. Gowda, D. C., Hogue-Angeletti, R., Margolis, R. K. and Margolis, R. U. (1990). Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A. Arch Biochem Biophys 281: 219-24. 44. Peterson, J. B., Nelson, D. L., Ling, E. and HoguiAngeletti, R. (1987). Chromogranin A like proteins in the secretory granules of a protozoan, Paramecium tatraurelia. Journal of Biological Chemistry, 262: 17264-7. 45. Wohlfarter, T., Fischer-Colbrie, R., Hogue-Angeletti, R., Eiden, L. E. and Winkler, H. (1988). Processing of chromogranin A within chromaffin granules starts at Cand N-terminal cleavage sites. Febs Letters 231: 67-70. 46. Cetin, Y., Muller-Koppel, L., Aunis, D., Bader, M. F. and Grube, D. (1989). Chromogranin A in the gastro-enteropancreatic system II. Chromogranin A in mammalian entero-endocrine cells. Histochemistry 92: 265-75. 47. Smith, A. D. and Winkler, H. (1967). Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla. Biochem J 103: 483-492. 48. Wu, H. J., Rozansky, D. J., Parmer, R. J., Gill, B. M. and O’Connor, D. T. (1991). Structure and function of the chromogranin A gene: clues to evolution and tissue-specific expression. J Biol Chem 266, 13130-13134. 49. Nakano, I., Funakoshi, A., Miyasaka, K., Ishida, K., Makk, G., Angwin, P., Chang, D. and Tatemoto, K. (1989). Isolation and characterization of bovine pancreastatin. Regul Peptides 25: 207-13. 50. Galindo, E., Rill, A., Bader, M-F. and Aunis, D. (1991). Chromostatin, a 20-amino acid peptide derived from chromogranin A, inhibits chromaffin cell secretion (1991). Proc Nat1 Acad Sci USA 88: 1426-30.