Proc. Natl. Acad. Sci. USA Vol. 86, pp. 1188-1192, February 1989 Biochemistry Expression of haptoglobin-related and its potential role as a tumor antigen (neoplasia/breast cancer/acute-phase ) FRANCIS P. KUHAJDA*, ASOKA I. KATUMULUWA, AND GARY R. PASTERNACK Department of Pathology, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205 Communicated by M. Daniel Lane, November 14, 1988 (receivedfor review August 3, 1988)

ABSTRACT These studies describe the detection of a pensity for tumor invasion and early metastasis. Clinically, haptoglobin species, its characterization as the HPR these properties manifested as a dramatically worsened product, and its association with both pregnancy and neopla- prognosis (7-9). In Western blots (immunoblots) of fractions sia. Previous work showed that the early recurrence of human of sera from pregnant women, this antiserum detected a set breast cancer correlated with immunohistochemical staining of proteins whose properties were inconsistent with those of with a commercial antiserum ostensibly directed against preg- PAPP-A, a homotetramer composed of 200-kDa polypep- nancy-associated plasma protein A (PAPP-A). Use of this tides. The studies below describe the purification of the antiserum to guide purification ofthe putative antigen led to the unexpectedly immunoreactive protein species from the present identification and purification of a strongly immuno- plasma ofpregnant women, its identification as the HPR gene reactive protein species distinct from PAPP-A that was present product, and its relationship to the clinically important in the plasma of pregnant women at term. Unlike PAPP-A, a antigen expressed in human breast carcinoma. homotetramer of 200-kDa polypeptides, the immunoreactive protein consists of a light (a) chain (16.5 kDa) and a heavy (J6) chain (40 kDa); protein microsequencing ofthe 1 chain showed MATERIALS AND METHODS it to be a member ofthe haptoglobin family. The a chain ofthis Patient Material. Term maternal plasma was obtained from haptoglobin species differs from ordinary haptoglobin 1 and 2 discarded EDTA-anticoagulated whole blood samples from a chains both structurally and immunologically and represents the delivery suite of The Johns Hopkins Hospital and stored the product of the HPR gene, haptoglobin-related protein at -70'C until use. Paraffin-embedded human breast carci- (Hpr), since (i) the apparent molecular mass is the same as that noma specimens were obtained from the Department of predicted for Hpr a chain, (ii) the peptide map differs from Pathology. that of haptoglobin 1 in a manner predicted by the HPR Electrophoresis and Western Blotting. NaDodSO4/poly- nucleotide sequence, (iii) monospecific that react acrylamide gel electrophoresis used the Laemmli system with epitopes shared by the unique a chain and a synthetic (10). For Western blotting, Laemmli gels were transferred to peptide derived from the HPR nucleotide sequence do not 0.45-,um nitrocellulose sheets (Schleicher & Schuell) in 96 detect these epitopes in either haptoglobin 1 or 2, and (iv) mM glycine/12.5 mM Tris/0.1% NaDodSO4/20% (vol/vol) sequences of a-chain peptides were consistent with this iden- methanol at 40 V, 250-300 mA, at 40C for 6 hr, evaluated for tification, excluding haptoglobin 1 but not haptoglobin 2. The transfer efficiency by Ponceau S staining, blocked with 3% immunohistochemical reactivity of antibodies raised to the (wt/vol) bovine serum , then incubated sequentially synthetic Hpr peptide is similar to that of anti-PAPP-A. with for 2 hr and with 1251-labeled protein A for 1 hr Moreover, staining of neoplastic breast tissue is abolished by with intervening Tris/saline washes (11). preincubation with purified Hpr. Protein Purification. Proteins reactive with IgGs in the whole IgG fraction ofa rabbit anti-PAPP-A antiserum (Dako, Santa Barbara, CA) were purified by successive chromato- The present studies describe the expression of haptoglobin- graphic steps monitored by OD280, analysis of fractions by related protein (Hpr), the product of the HPR gene, and its Coomassie blue-stained Laemmli gels, and Western blotting. association with both pregnancy and neoplasia. Conventional Initially, 80 ml of plasma was loaded onto a 2.5 x 30 cm haptoglobins (Hps) have been well studied; the three known Cibacron Blue F3G-A Sepharose (Pharmacia) column equil- each code for a precursor containing both the a and p8 ibrated in 50 mM sodium phosphate (pH 6.8) at 40C. The chains (1-3). Maeda (4) described the HPR as a stretch flow-through containing detectable immunoreactive material of DNA located 2.2 kilobases downstream from the conven- was dialyzed against 20 mM sodium phosphate (pH 6.8 at tional HP locus. This sequence appears to be an intact gene 4°C) with 1 mM NaN3 and applied to a 2.5 x 10 cm column coding for a theoretical protein whose a and f8 chains are of DEAE-cellulose (Pierce) equilibrated in the same buffer. distinct from, but highly homologous to, conventional Hps, The immunoreactive material was eluted with 0.2 M NaCl in particularly haptoglobin 1 (Hp-1). Nevertheless, several the starting buffer, dialyzed against 20 mM Tris HCl (pH 8.5 investigators failed to detect Hpr expression (4-6). Interest- at 4°C) containing 0.15 M NaCl, 1 mM 2-mercaptoethanol, ingly, the locus contains a retrovirus-like element and is and 1 mM NaN3, then loaded onto a 2.5 x 60 cm column of duplicated in some individuals (4, 5). Fast Flow Q-Sepharose (Pharmacia) equilibrated in the same Experiments directly leading to the present studies found buffer. The column was eluted with a 500-ml linear gradient that a commercial antiserum to pregnancy-associated plasma of 150-500 mM NaCl at a flow rate of 100 ml/hr at 4°C. The protein A (PAPP-A) reacted immunohistochemically with immunoreactive proteins eluted between 0.25 and 0.28 M human breast carcinomas and that those breast carcinomas NaCl. Fractions containing immunoreactive protein were staining positively for the antigen showed an increased pro- Abbreviations: Hp, haptoglobin; Hpr, haptoglobin-related protein; The publication costs of this article were defrayed in part by page charge PAPP-A, pregnancy-associated plasma protein A; KLH, keyhole payment. This article must therefore be hereby marked "advertisement" limpet hemocyanin; CEA, carcinoembryonic antigen. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1188 Downloaded by guest on October 2, 2021 Biochemistry: Kuhajda et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1189 pooled, dialyzed against 200 mM Tris acetate (pH 7.5 at 40C) Affinity Chromatography. Hp-i and the Hpr synthetic with 1 mM 2-mercaptoethanol/1 mM NaN3, and applied to a peptide were immobilized on agarose beads derivatized with 5 x 90 cm Sepharose CL4B (Pharmacia) column. To separate 1,1'-carbonyldiimidazole [Reacti-Gel (6X), Pierce], which the individual immunoreactive chains under denaturing con- results in a stable N-alkylcarbamate linkage. Gel (5 ml) was ditions, fractions containing immunoreactive protein were washed with 0.1 M borate/0.15 M NaCl, pH 8.5, at room pooled and dialyzed against 20 mM Tris HCI, pH 8.5/6 M temperature to remove the acetone. Hp-1 (4 mg) (Sigma) urea/10 mM 2-mercaptoethanol, followed by HPLC on a diluted to 1 mg/ml in borate buffer or 50 mg of Hpr synthetic Waters system with a 5 x 50 mm Mono Q HR5/5 (Pharmacia) peptide diluted to 12.5 mg/ml in borate buffer was added to column monitoring OD280. The sample was injected in starting 5 ml ofgel and incubated with agitation at 40C for 30 hr. After buffer, a 10-ml wash followed, and a linear gradient of 30 ml incubation, the supernatant was decanted and an equal over 15 min to final conditions of 0.5 M added NaCl was volume of 0.2 M Tris was added to quench any remaining applied. The immunoreactive 40-kDa protein was eluted in reactivity. The gels were extensively washed with Tris/saline =0.85 M NaCl. buffer. The Hpr peptide column was then denatured with 100 Amino Acid Sequencing. Protein chains purified by anion- ml of 0.1% NaDodSO4 in Tris/saline buffer. exchange HPLC were homogenous on Laemmli gels. For Monospecific anti-Hpr antibodies were isolated from the sequencing, samples were dialyzed against three changes of crude anti-PAPP-A serum by sequential affinity chromato- 0.2 M NH4HCO3 and thrice lyophilized from HPLC-grade graphic steps. First, 100 A.l ofcrude anti-PAPP-A antibody in water. Gas-phase sequencing was carried out on an Applied 10 ml of Tris/saline buffer was incubated with the Hp-1 gel Biosystems model 470A sequencer by P. Shenbagamurthi overnight at 40C with agitation. The gel was next washed (Protein Chemistry Facility, The Johns Hopkins University extensively with Tris/saline and the flow-through was col- School of Medicine). Samples of intact protein chains gen- lected over a 5-ml hydroxyapatite column. Antibodies that erally consisted of 100 ,ug of protein by Lowry assay (12) bound to the Hp-1 column were eluted with 20 ml of acetate 0.5-2 nmol as buffer (0.05 M sodium acetate/0.15 M NaCl, pH 3.5, at room yielding quantitated by the sequencer. temperature). The antibodies that flowed through the column Peptide Mapping. Peptide mapping was performed using the Elder were eluted from the hydroxyapatite column with 20 ml of0.4 technique (13, 14). M sodium phosphate. Both sets of antibodies were dialyzed Preparation of Anti-Hpr Antibody. A synthetic peptide against 2 liters of Tris/saline buffer. corresponding to the 34 N-terminal residues of the predicted The antibodies that did not bind to the Hp-i column were HPR gene product (4) was synthesized by the Protein Chemis- then incubated with the denatured Hpr peptide gel overnight try Facility on an Applied Biosystems model 43A peptide at 4°C with agitation. Antibodies that bound to the Hpr synthesizer (15). Because histidine coupling is inefficient, peptide were eluted with 20 ml of acetate buffer and dialyzed lysine was conservatively substituted for the histidine in against Tris/saline. position 28. For immunization, the peptide was conjugated to Immunohistochemistry. Immunohistochemistry was per- keyhole limpet hemocyanin (KLH) (Boehringer Mannheim) formed using the unlabeled antibody immunoperoxidase essentially as described (16). The KLH-maleimidobenzoyl- technique for formalin-fixed paraffin-embedded tissues (7). n-hydroxysuccinimide ester (MBS) conjugate was separated from unreacted MBS by gel filtration on a Pharmacia PD-10 column. Hpr peptide (1.2 mg) was added directly to the RESULTS KLH-MBS and the mixture was incubated a further 30 min Purification of Immunoreactive Proteins from Pregnancy at room temperature. Coupling efficiency was estimated Serum. The initial goal was to characterize the substance in using Pharmacia Superose 12 FPLC by peak integration and human breast carcinoma that reacted with the crude IgG comparison to standards of peptide run alone. The KLH- fraction of a commercially produced antiserum to PAPP-A. peptide was separated from unreacted peptide by gel filtra- The antiserum both guided the purification ofthe homologous tion in isotonic phosphate-buffered saline (PBS) on a PD-10 antigen from pregnancy serum and helped characterize the column. This method yielded an approximate substitution immunoreactive material in breast cancer. PAPP-A is a ratio of 4.6 peptides per KLH molecule. homotetramer of 200-kDa polypeptides (17); surprisingly, Two Pasteurella-free New Zealand White rabbits (Ha- anti-PAPP-A failed to detect such a protein in Western blots zelton Research Products, Reston, VA) were each inoculated of pregnancy serum or various subfractions. Instead, three on day 0 with 200 ug of antigen in 1 ml of an emulsion immunoreactive chains were consistently seen with apparent comprised of equal parts of PBS and complete Freund's molecular masses of 40 kDa, 20 kDa, and 16.5 kDa in adjuvant; the inoculae were divided among three or four Laemmli gels, the 16.5-kDa species reacting most strongly subcutaneous and intramuscular sites. On day 14, the animals and the 20 kDa reacting most weakly. Reactivity with several were given booster inoculations with similar amounts of unidentified protein species and the undetectable immuno- antigen in incomplete Freund's adjuvant. The rabbits were reactivity with PAPP-A in Western blots suggested that one test-bled on day 32; serum from the positive rabbit was or more of the unidentified bands might be related to the collected over the ensuing month. immunoreactive material in neoplastic breast tissue. To purify the antibody, a 10 cm x 5 mm SelectiSpher-10 Fig. 1 summarizes the purification of the unidentified activated-tresyl HPLC affinity column (Pierce) was deriva- immunoreactive bands. The figure shows a Coomassie blue- tized with the Hpr synthetic peptide. The column was washed stained 10-15% polyacrylamide Laemmli gel and corre- with 30 ml of PBS at 2 ml/min, then 27 mg of peptide sponding Western blot. Sequential chromatographic steps dissolved in 4.5 ml of PBS was applied at a rate of 1 ml/min. involved removal of albumin and additional protein species Unreacted tresyl groups were capped with 30 ml of 0.2 M on Cibacron Blue F3GA Sepharose (lanes a), step elution Tris HCI (pH 8.0) through the column. For antibody purifi- from a DEAE-cellulose column (lanes b), gradient elution cation, the column was equilibrated in 300 mM NaCI/20 mM from a Fast-Flow Q Sepharose anion-exchange column (lanes sodium phosphate, pH 7.5. Serum (10-20 ml) was loaded at c), and gel permeation chromatography on Sepharose CL4B 0.5 ml/min. After loading, the column was washed with (lanes d). Interestingly, gel filtration incompletely resolved buffer until the 0D280 of the effluent returned to zero. the immunoreactive proteins into three consecutively eluting Antibody was eluted with 6 M urea in the same buffer, and species consisting of the 40-kDa species co-eluting with the the antibody-containing fractions were immediately dialyzed weakly reactive 20-kDa species, the 40-kDa band co-eluting into Tris-buffered saline with 1 mM NaN3. with a weakly immunoreactive 16.5-kDa species, and lastly, Downloaded by guest on October 2, 2021 1190 Biochemistry: Kuhajda et al. Proc. Natl. Acad. Sci. USA 86 (1989)

a b c d a b c d ab c a b c a b c

kDa

40-- *"_,xs. * I 0',

aw 4NOW .4 20-- 1 6.5

_A B C FIG. 1. Purification of Hpr. A 10-15% polyacrylamide gradient Laemmli gel and corresponding Western blot of sequential chro- FIG. 2. Immunologic analysis of Hp structure. (A) Coomassie matographic purification ofplasma proteins reactive to anti-PAPP-A blue-stained 10-15% polyacrylamide gradient gel of Hp-1, Hp-2, and are shown. (Left) Coomassie blue-stained gradient gel. (Right) purified pregnancy-associated Hp in lanes a-c, respectively. (B) Immunoblot incubated with 25 ml of anti-PAPP-A, diluted 1:50. Corresponding immunoblot with anti-Hp diluted 1:50. (C) Immuno- Lanes: a, Cibacron Blue Sepharose flow-through; b, a 0.2 M NaCl blot with anti-PAPP-A diluted 1:50. Anti-Hp reacts strongly with all DEAE-cellulose step elution; c, fraction from Fast-Flow Q Sepha- the 40-kDa P chains, and Hp-1 and -2 a chains; the 16.5-kDa band c rose anion exchange; d, Sepharose CL-4B fraction. Note the suc- from lane containing the purified protein is only weakly reactive. cessive enrichment and co-elution of the strongly immunoreactive In contrast, anti-PAPP-A, while strongly labeling the 40-kDa f8 40-kDa and 16.5-kDa bands and weakly immunoreactive 20-kDa chains, reacts weakly with Hp-2 a chain and is only slightly reactive species. with Hp-1 a chain. The 16.5-kDa band from the purified protein in lane C is now strongly reactive. The pregnancy-associated Hp differs from Hp-1 and -2 in its 16.5-kDa a chain. as a separate, albeit overlapping peak, the 40-kDa species co-eluting with a strongly immunoreactive 16.5-kDa band. and distribution, recognizing all three Hp chains; the relative The relationship between the purified protein and the immunoreactivity of the 16.5-kDa chain of the purified immunoreactive material in breast cancer was explored using protein is commensurate with the relative intensity of its a human breast carcinoma known to stain positively both Coomassie blue staining. Anti-PAPP-A also reacts with Hp with anti-PAPP-A and with anti-carcinoembryonic antigen heavy chains and Hp-2 light chain; however, its relative (CEA). Preincubation of anti-PAPP-A with a mixture of the immunoreactivity with the 16.5-kDa chain from pregnancy purified protein species ablated immunostaining, but prein- serum shown in lane c is disproportionately intense in cubation of anti-CEA under similar conditions had no effect, comparison to its Coomassie blue staining, particularly when thus demonstrating an immunologic relationship between the compared to the disproportionately weak relative immuno- material present in breast cancer and that found in pregnancy reactivity of the Hp-1 a chain shown in lane a. Thus, the a serum (data not shown). chain isolated from pregnancy plasma is immunologically Identification as a Hp by Protein Microsequencing. The distinct, containing epitopes seen by the anti-PAPP-A that 40-kDa chain was isolated by anion-exchange HPLC under are not present in the a chains of either Hp-1 or Hp-2. denaturing conditions. N-terminal sequencing yielded 20 In addition to immunologic analysis, peptide mapping (13, residues. Using the protein sequence data base of the 14) was performed to identify potential structural differences National Biomedical Research Foundation (November 1987) between heavy and light chains from the Hps and purified and the DFASTP alignment program, three protein sequences pregnancy protein. Peptide mapping by this technique de- showed 100% homology over the entire 20-amino acid stretch tects only tyrosine-containing peptides. Using the published with the chain of human Hp-i, the 8 chain of human amino acid sequences, the family oftryptic peptides from the haptoglobin 2 (Hp-2), and the predicted ,8 chain of the Hpr a chains of Hp-1 and Hp-2 should each be similar since Hp-2 precursor (6, 18-21). Hp molecules are composed of light (a) resulted from a partial reduplication of Hp-1 (1) (Fig. 3 A and and heavy (,8) chains synthesized as a single chain and then C). The principal differences in amino acid sequence of Hpr, cleaved to form the final product (22). Hps migrate aberrantly Hp-1, and Hp-2 lie in the a chains. Within the a chains, these slowly on Laemmli gels with apparent molecular masses of differences cluster principally at the N termini. The deduced 39.5 kDa for the glycosylated chains and of 16.5 kDa for the Hpr a-chain sequence predicts the same tryptic peptides as carbohydrate-free a chain of Hp-1 and 20 kDa for the a chain obtained from Hp-1 plus one additional tyrosine-containing of Hp-2 (1). The molecular masses found by gel electropho- peptide. Fig. 3B shows the peptide map ofthe purified a chain resis for both the heavy and light chains ofthe purified protein from the pregnancy-associated protein with an arrow high- are similar to those reported for Hp-1. lighting the one additional peptide obtained experimentally. Hp Purified from Pregnancy Plasma Possesses Distinctive These results thus raise the possibility that the Hp purified Structural Features. Although sequence analysis placed the from pregnancy plasma might represent the HPR gene pregnancy plasma protein unequivocally in the HP gene product. Fig. 3D is a mixing experiment where Hp-1 and the family, more specific assignments could not be made, since purified pregnancy-associated protein a chain were mixed the N-terminal sequence for the 8 chains of all species is before mapping. Again, the additional peptide is present but identical. Fig. 2 shows the results of an immunologic analysis with less relative intensity, while the shared peptides all performed with purified Hp-i and -2 standards, the Hp co-migrate. purified from pregnancy serum, rabbit anti-Hp, and anti- Hp a Chain from Pregnancy Serum Contains Hpr Epitopes. PAPP-A. Fig. 2A shows a 10-15% polyacrylamide Coomassie If the immunologically and structurally distinct Hp from blue-stained Laemmli gel of Hp-i, Hp-2, and the Hp purified pregnancy serum is the HPR gene product, then its a chain from pregnancy serum. The corresponding Western blot with should share unique epitopes with a synthetic peptide derived anti-Hp mimics the Coomassie blue-stained gel in its intensity from the deduced Hpr sequence. A 34-amino acid peptide Downloaded by guest on October 2, 2021 Biochemistry: Kuhajda et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1191

.A! "lmwB intense labeling of the 16.5-kDa pregnancy-associated Hp a chain along with the 40-kDa ,1 chains. ... X~ 4 C and D show the results of ofthe ..~~~~~f i la Fig. immunoabsorption wg anti-PAPP-A antibody on immobilized Hp-1. The blot in C was incubated with the antibodies reactive to were b.-.1~A Hp-i that _ __D eluted from the column, while the blot in D was incubated with antibodies in the flow-through. Antibodies reactive to the a chains of pregnancy-associated Hp and Hp-2 flowed through the Hp-1 column, whereas antibodies against the 13 chains were retarded. The antibod- *S flow-through containing ies specific for the a chain of the pregnancy-associated Hp were then incubated with immobilized synthetic Hprpeptide. FIG. 3. Tryptic peptide maps of Hp a chains. (A) Hp-1 a chain. The positively selected antibodies binding to the Hpr peptide (B) Pregnancy-associated Hp a chain. (C) Hp-2 a chain. (D) Mixing were eluted and incubated with the blot in E. Only the a chain experiment with Hp-1 and purified immunoreactive a chains. Arrow of the pregnancy-associated Hp reacted with antibodies indicates tryptic peptide. Since one additional tryptic peptide is specific for the Hpr synthetic peptide. This demonstrates that predicted from the amino acid sequence of the Hpr a chain, this the a chain of pregnancy-associated Hp shares epitopes with evidence suggests that the pregnancy-associated Hp is the product of the Hpr synthetic peptide. the HPR gene. Hpr Epitopes Identified in the Cytoplasm of Human Breast Carcinoma. An immunoperoxidase staining and absorption was synthesized that was identical to the predicted N experiment was performed with anti-Hpr, anti-PAPP-A, and terminus of Hpr a chain except for one conservative amino purified Hpr. If the immunoreactivity of human breast acid substitution. This peptide was immobilized on agarose carcinoma with the anti-PAPP-A antibody was due to Hpr, beads for immunoabsorption in conjunction with separate preincubation ofanti-PAPP-A with native Hpr should abolish columns of Hp-i and Hp-2. Thus, antibodies reacting with the immunoreactivity. Similarly, neoplastic breast tissue that epitopes shared with the conventional Hps should bind to the stains with anti-PAPP-A should stain with anti-Hpr, with the Hp columns, and antibodies reacting with the synthetic same pattern of competition. peptide could be positively selected. When anti-Hpr antibody was incubated for 2 hr at 40C with Fig. 4 shows, by stepwise immunoselection, that the native Hpr, cytoplasmic staining was abolished (Fig. 5). To antibodies that react to the a chain ofthe Hp from pregnancy establish specificity of the immunoabsorption, when anti- serum also react with the Hpr synthetic peptide. Fig. 4A is a CEA antibodies were incubated under similar conditions with 10-15% polyacrylamide gradient Laemmli gel with Hp-i in native Hpr, anti-CEA cytoplasmic staining was not abol- the left lane and purified pregnancy-associated Hp in the right ished. This established that the substance identified by lane; B-E are immunoblots of these same proteins. Fig. 4B anti-PAPP-A in human breast carcinoma expresses epitopes was incubated with unabsorbed anti-PAPP-A serum, and, as derived from the N terminus of the predicted Hpr a chain. in Fig. 2C, there is scant reactivity to the Hp-1 a chain, weak This antigen must, therefore, represent Hpr, a modified form reactivity to the contaminating 20-kDa Hp-2 a chain, but of Hpr, or a highly cross-reactive protein.

A B C D E DISCUSSION These studies describe the expression of a protein product derived from the human HPR locus, a gene for a Hp located jI 2.2 kilobase pairs downstream from the conventional HP kDa locus (5). The following several pieces of evidence support the hypothesis that the protein isolated from pregnancy 40 _t__,_ plasma is Hpr: (i) affinity-purified antibodies reactive to the a chain of the purified protein also react with a synthetic peptide representing the 34 N-terminal amino acids of the predicted Hpr a chain, but not with the a chains from Hp-i or -2; (ii) the molecular mass of the a chain of the purified 4mm' protein is consistent with that predicted for the Hpr a chain,

FIG. 4. The 16.5-kDa a chain of the purified immunoreactive protein contains Hpr epitopes. (A) Coomassie blue-stained 10-15% polyacrylamide gradient Laemmli gel with 25 jug of Hp-1 in the left lane and 100 ,ug of pregnancy-associated Hp in the right lane. (B-E) a to Immunoblots of the same protein. The blot in B was incubated with AA ZB unabsorbed __w.mww anti-PAPP-A antibody diluted 1:50. The blot in C represents the subset of anti-PAPP-A antibodies that bound to immobilized Hp-1; the blot in D was incubated with the unreactive antibodies that flowed through. Note the enrichment for antibodies to the 40-kDa f8 chains that bound to the Hp-1 gel in C, while antibodies to the immunoreactive a chain were unreactive with FIG. 5. Immunoperoxidase staining with anti-Hpr synthetic pep- immobilized Hp-1 and flowed through (D). The blot in E represents tide antibody in human breast cancer. (A) Strong cytoplasmic the antibodies seen in D that reacted with immobilized, denatured, staining with anti-Hpr is shown that is abolished by preincubation synthetic Hpr peptide. Note that these antibodies, which bound to with the synthetic purified immunoreactive protein (B). (C) Anti-CEA staining is Hpr peptide, react only with the 16.5-kDa a chain ofthe shown that is not absorbed by preincubation with immunoreactive pregnancy-associated Hp. This demonstrates that the a chain of this protein (D). Thus Hpr, or an immunologically similar substance, is Hp contains Hpr epitopes. present in human breast carcinoma. Downloaded by guest on October 2, 2021 1192 Biochemistry: Kuhajda et al. Proc. Natl. Acad Sci. USA 86 (1989) co-migrating on Laemmli gels with the Hp-1 a chain; (iii) the might be responsible for Hpr production during pregnancy. In peptide map of the purified a chain differs from the maps of breast cancer, malignant cells might elaborate Hpr them- the a chains of Hp-1 and Hp-2 in the manner that would be selves, or internalize it by way of a surface receptor. predicted from the Hpr a-chain sequence; and (iv) the Hpr a Although the granular cytoplasmic staining for Hpr suggests chain co-purifies with a second polypeptide chain whose size synthesis and secretion by the breast carcinoma cells, the and N-terminal amino acid sequence are consistent with answer must await biosynthetic studies. those ofHp p chains. Additional support comes from sequenc- The isolation of Hpr from pregnancy plasma and the ing. Unfortunately, the N terminus of the purified Hpr a chain localization of immunologically related material, if not Hpr is blocked, and attempts to isolate enzymatically cleaved itself, in virulent breast carcinoma broadens the spectrum of peptides from the N-terminal portion of the molecule were pathophysiologic roles of Hps. Detection and quantitation of unsuccessful. Two peptides, Gly-Lys-Pro-Lys-Asn-Pro-Ala- Hpr may prove to be a useful diagnostic procedure in cancer Asn-Gln-Val-Gln-Arg and Thr-Leu-Asn-Asp-Lys-Lys-Glu- and, possibly, pregnancy (35). As the comparison between Trp, were isolated whose sequences are common to Hpr and neoplastic states and pregnancy is often drawn, Hpr may Hp-2 a chains, but are not contained in the sequence of the subsume similar functions in pregnancy and neoplasia, aiding Hp-1 a chain. In view of the other data, including high molar development in one instance while facilitating tumor invasion yields of these peptides and the absence of any detectable and metastasis in the other. contaminating Hp-2, it is most likely that these peptides are derived from the a chain of Hpr. This work was supported by U.S. Public Health Service Grants their R01 CA 46143 and R01 GM 36697, a career development grant from Hps are acute-phase proteins initially discovered by the Andrew W. Mellon Foundation (G.R.P.), Career Development ability to bind , enhancing the endogenous per- Award 27-85 from the American Cancer Society (F.P.K.), and by oxidase activity of hemoglobin (1-3). Traditionally, Hp National Institutes of Health Training Grant T32 HL 07525 (A.I.K.). function has been thought principally to be iron conservation mediated by its ability to bind hemoglobin. Hps would thus 1. Bowman, B. H. & Kurosky, A. (1982) Adv. Hum. Genet. 12, 189-261. prevent renal excretion of free hemoglobin and recycle iron 2. Javid, J. (1978) Curr. Top. Hematol. 1, 151-192. 3. Sutton, H. E. (1970) Prog. Med. Genet. 7, 163-216. to the by a hepatocyte cell surface receptor for the Hp- 4. Maeda, N. (1985) J. Biol. Chem. 260, 6698-6709. hemoglobin complex (23-26); additionally, by binding iron, 5. Maeda, N., McEvoy, S. M., Harris, H. F., Huisman, T. H. & Smithies, they might also exert an antibacterial effect (27). The present 0. (1986) Proc. Natl. Acad. Sci. USA 83, 7395-7399. in 6. Bensi, G., Raugei, G., Klefenz, H. & Cortese, R. (1985) EMBOJ. 4, 119- data suggest additional possibilities. The detection of Hpr 126. maternal serum suggests that Hpr may play a role in preg- 7. Kuhajda, F. P. & Eggleston, J. C. (1985) Am. J. Pathol. 121, 342-348. nancy. Unless it represents a fetal Hp that crossed the pla- 8. Kuhajda, F. P., Abeloff, M. D. & Eggleston, J. C. (1985) Hum. Pathol. centa, the Hpr purified from maternal serum suggests maternal 16, 228-235. 9. Kuhajda, F. P. & Eggleston, J. C. (1985) Lab. Invest. 53, 101-107. expression during pregnancy to carry out a yet unknown 10. Laemmli, U. K. (1970) Nature (London) 227, 680-685. function. 11. Gershoni, J. M. & Palade, G. E. (1983) Anal. Biochem. 131, 1-15. In addition to pregnancy plasma, a species reacting with 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951)J. was detected immunohistochem- Biol. Chem. 193, 265-275. anti-Hpr peptide antibody 13. Speicher, D. W. Morrow, J. S., Knowles, W. J. & Marchesi, V. T. ically in human breast carcinoma. The anti-Hpr synthetic (1980) Proc. Natl. Acad. Sci. USA 77, 5673-5677. peptide antibody showed a cytoplasmic staining pattern 14. Elder, J. H., Pickett, R. A., II, Hampton, J. & Lemer, R. A. (1977) J. similar to the commercial anti-PAPP-A antibody used in Biol. Chem. 252, 6510-6515. was 15. Merrifield, R. B. (1968) J. Am. Chem. Soc. 85, 2149-2154. previous studies (7-9). In both cases, positive staining 16. Lerner, R. A., Green, N., Alexander, H., Liu, F. T., Sutcliffe, J. G. & abolished when the antibody was preincubated with purified Shinnick, T. M. (1981) Proc. Natl. Acad. Sci. USA 78, 3403-3407. Hprj whereas neither Hp-1 nor Hp-2 had any effect, thus 17. Bohn, H. (1985) in Proteins of the Placenta, eds. Bischof, P. J. & that is present in the of Klopper, H. (Karger, Basel), pp. 1-25. strongly inferring Hpr cytoplasm 18. Kurosky, A., Barnett, D. R., Lee, T. H., Touchstone, B., Hay, R. E., breast cancer cells. The immunoreactivity with the anti-Hpr Arnott, M. S., Bowman, B. H. & Fitch, W. M. (1980) Proc. Nat!. Acad. synthetic peptide antibody, like the anti-PAPP-A antibody, Sci. USA 77, 3388-3392. distinguished a group of patients with a dramatically wors- 19. Black, J. A. & Dixon, G. H. (1968) Nature (London) 218, 736-741. and The 20. Black, J. A., Chan, G. F., Hew, C. L. & Dixon, G. H. (1970) Can. J. ened prognosis (F.P.K. G.R.P., unpublished data). Biochem. 48, 123-132. finding of Hpr immunoreactivity within cancer cells, the 21. Black, J. A. & Dixon, G. H. (1970) Can. J. Biochem. 48, 133-146. propensity to localize in invasive, rather than in situ tumors, 22. Haugen, T. H., Hanley, J. M. & Heath, E. C. (1981) J. Biol. Chem. 256, and its consistent association with phenotypically aggressive 1055-1057. that or related molecules serve 23. Limet, J. N., Quintart, J., Otte-Slachmuylder, C. & Schneider, Y. J. neoplasia suggests Hpr may (1982) Acta Biol. Med. Ger. 41, 113-124. as a mediator of certain malignant processes. Although the 24. Kin&, K., Tsunoo, H., Higa, Y., Takami, M. & Nakajima, H. (1982) J. mechanism whereby Hpr might influence the behavior of a Biol. Chem. 257, 4828-4833. malignancy is unclear, previous studies reported unusual 25. Kino, K., Tsunoo, H., Higa, Y., Takami, M., Hamaguchi, H. & of which modulate Nakajima, H. (1980) J. Biol. Chem. 255, 9616-9620. Hps, some lymphocyte responsiveness 26. Higa, Y., Oshiro, S., Kino, K., Tsunoo, H. & Nakajima, H. (1981)J. Biol. (28-30). In all cases, such Hps were detected in abnormal Chem. 256, 12322-12328. body fluids whose accumulation was directly due to a 27. Eaton, J. W., Brandt, P., Mahoney, J. R. & Lee, J. T. (1982) Science malignancy; however, none has been precisely character- 215, 691-693. that modulate the host 28. Samak, R., Edelstein, R. & Israel, L. (1982) Cancer Immunol. Immu- ized. It is thus possible Hpr might nother. 13, 38-43. response to a malignancy so as to enable it to survive and 29. Israel, L., Samak, R., Edelstein, R., Bogucki, D. & Breau, J. L. (1981) expand clonally. In addition, Hps from cancerous body fluids Ann. Med. Interne 132, 26-29. have been shown to stimulate collagen synthesis in fibro- 30. Baseler, M. W. & Burrell, R. (1983) Inflammation 7, 387-400. interface between tumor 31. Borel, J. P., Viellard, A. & Randoux, A. (1976) Agents Actions 6, 207- blasts (31, 32), suggesting that the 211. cells and the extracellular matrix might be another locus of 32. Viellard, A., Lonbart, C., Borel, J. P. & Jayle, M. F. (1974) Pathol.-Biol. action. 22, 741-742. While no expression of the HPR gene has been identified 33. Wada, T., Oara, H., Wanatabe, K., Kinoshita, H. & Nishio, H. (1970) J. or fetal mRNA Reticuloendothel. Soc. 8, 195-207. in adult hepatocyte (5, 6), Hp synthesis may 34. Dziegielewska, K. M., Saunders, N. R., Schejter, E. J., Zakut, H., not be restricted to the liver. In some instances, lymphocyte Zevin-Sonkin, D., Zisling, R. & Soreq, H. (1986) Dev. Biol. 115, 93-104. cultures (33) and brain (34) synthesize tips, suggesting that an 35. Bischof, P., Haenggeli, M. T., Sizonenko, M. T., Herrmann, W. & alternative site of synthesis, such as placenta or decidua, Sizonenko, P. C. (1981) Biol. Reprod. 24, 1076-1081. Downloaded by guest on October 2, 2021