polypeptide core of a human pancreatic tumor mucin antigen

1990;50:2997-3001.Cancer Res Michael S. Lan, Michael A. Hollingsworth and Richard S. Metzgar Polypeptide Core of a Human Pancreatic Tumor Mucin Antigen

Updated Version http://cancerres.aacrjournals.org/content/50/10/2997

Access the most recent version of this article at:

Citing Articles http://cancerres.aacrjournals.org/content/50/10/2997#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications

[email protected] at

To request permission to re-use all or part of this article, contact the AACR Publications

American Association for Cancer Research Copyright © 1990 on February 20, 2013cancerres.aacrjournals.orgDownloaded from

http://cancerres.aacrjournals.org/content/50/10/2997

http://cancerres.aacrjournals.org/content/50/10/2997#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]

http://cancerres.aacrjournals.org/

http://www.aacr.org/

(CANCER RESEARCH 50. 2997-3001. May 15, 1990]

Polypeptide Core of a Human Pancreatic Tumor Mucin Antigen1

Michael S. Lan,2 Michael A. Hollingsworth, and Richard S. Metzgar3

Department of Microbiology and Immunology, Duke L'niversity Medical Center, Durham, North Carolina 27710

ABSTRACT

This work describes the molecular properties of the polypeptide coreof a human pancreatic mucin antigen isolated from a human pancreaticadenocarcinoma cell line, HPAF. Pancreatic tumor mucin was isolatedby a combination of molecular sieve Chromatograph) and CsCI/4 Mguanidine-HCI density gradient ultracentrifugation. Trifluoromethanesulfonic acid was used to remove carbohydrate units from purified mucinmolecules. A rabbit monospecific polyclonal antibody was generatedagainst pancreatic apomucin and reacted with a M, > 200,000 species.The antibody binding data indicated that the rabbit antiserum raisedagainst pancreatic apomucin cross-reacted with a breast mucin syntheticpeptide. Northern blot and immunodot blot analyses of various cell lineextracts revealed that a tandem repeat sequence and a similar mRNAwere detected in both pancreatic and breast mucin-producing cell lines.These results suggest that pancreatic apomucin and breast apomucinshare some similarity in tandem repeated nucleic acid and protein sequences.

INTRODUCTION

Mucins and high-molecular-weight glycoproteins have oftenbeen implicated as tumor-associated antigens of adenocarcino-mas from a variety of organ and tissue sites. Although manymonoclonal antibodies have recognized the sugar moieties ofmucins and demonstrated tumor specificity (1), biochemicaldefinition of antigenic determinants on these molecules wasalways hampered by the complexity of carbohydrate side chains.Many reports have studied the biochemical properties of variousmucin-type tumor-associated antigens (2-5). It has becomeobvious that these mucin-type antigens have very distinct carbohydrate compositions. Although they have all demonstratedcharacteristic mucin-type amino acid compositions and molecular properties, it has been difficult to compare the molecularstructures of the polypeptide core of mucin molecules derivedfrom different organ sites. Recent studies with a monoclonalantibody, SM3, raised to a breast mucin peptide indicated thatthis antibody was useful in detecting benign from malignantbreast disease (6). Therefore, in addition to carbohydrate determinants, it is important to characterize the molecular structureof mucin polypeptide backbones from other organ sites and toevaluate their relationships to transformation and differentiation of human tumors. In this study, a purified pancreatic tumormucin was subjected to TFMS4 deglycosylation (7). Rabbit

polyclonal antiserum was generated to detect and to characterize the mucin polypeptide core. Data from antibody bindingassays suggested that pancreatic apomucins share some similarity with breast apomucins. This result was supported byNorthern blot and immunodot blot analyses which revealed

Received 6/22/89; revised 2/9/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This research was supported by Grants CA 40044 and CA 32672 from the

National Cancer Institute.2Recipient of a postdoctoral fellowship from the National Cancer Center.J To whom requests for reprints should be addressed.4The abbreviations used are: TFMS. trifluoromethane sulfonic acid; HPAF,

pancreatic cell line derived from human pancreatic ascitic fluid; SDS. sodiumdodecyl sulfate; PAGE, polyacrylamide gel electrophoresis: GlcNAc, /V-acetylglu-cosamine; GalNAc, A'-acetylgalactosamine; PBS, phosphate-buffered saline;cDNA, complementary DNA.

that similar messages and precursors were detected in variouspancreatic and breast mucin-producing carcinoma cell lines.

MATERIALS AND METHODS

Cell Lines and Culture Conditions. The tumor cell lines used wereobtained from the following sources: Panc-1, Hs766T, Aspc-1, Capan-1, BxPc-3, BT-20, MCF-7, SKBR-3, and LS-180 from the AmericanType Culture Collection; T3M4 from T. Okabe (Tokyo); Colo 357from George Moore (Denver, CO); SW979 from H. Kalthoff (Hamburg, Federal Republic of Germany); SKMEL and DM-6 from H.Seigler(Duke University, Durham, NC); and RW 7213 from L.Tibbetts(Providence, RI). The HPAF cell line was established in our laboratory(8). Tumor cell lines were cultured in minimal essential medium supplemented with 10% fetal calf serum.

Purification of Pancreatic Tumor Mucin from a Pancreatic Adenocarcinoma Cell I.ine (HPAF). The purification scheme of pancreatic tumormucins involved a slight modification of previously established procedures (9). Briefly, HPAF tissue culture spent medium was concentratedby 50 to 75% ammonium sulfate fractionation. The concentrated sample was subjected to a Sepharose CL-4B column (2.5 x 95 cm) whichwas preequilibrated with 0.1 Mammonium acetate buffer in the presenceof 0.1% SDS and l mM dithiothreitol. The mucin molecule wasexcluded from the Sepharose CL-4B column which demonstrated amolecular mass above 1 x IO6 daltons. The excluded fractions were

dialyzed against 8 M urea and then PBS to remove SDS detergent.Molecular sieve-purified mucin antigen was added to 40% (w/w) CsClin the presence of 4 M guanidine-HCI as described by Carlstedt et al.(10) at a loading density of 1.42 g/ml. Gradients were formed bycentrifugation in a Beckman Model VTi 65.1 vertical rotor at 55,000rpm for 48 h at 20Â°C.Fractions were collected, and densities were

determined by weighing a 100-^1 aliquot of each fraction. The purity ofthe fractions was monitored by SDS-PAGE and silver staining.

Deglycosylation of Pancreatic Tumor Mucins by TFMS Treatment.Deglycosylation of mucin glycoproteins with TFMS was performedaccording to the procedure of Edge et al. (7). One part of anisÃ³leandtwo parts of TFMS (Aldrich Chemical Co.. Milwaukee, WI) were mixedand cooled to 0Â°C,and then purified pancreatic mucins (1 to 5 mg)

were dissolved in 1 ml of this mixture in a reaction vial (Pierce ChemicalCo., Rockford, IL) under nitrogen. The reaction was performed at roomtemperature for 3 h. The TFMS-deglycosylated mucins were neutralized with an equal volume of ice-cold 50% (v/v) aqueous pyridine. Etherextraction of the aqueous phase was repeated several times, and thefinal products in aqueous phase were dialyzed against several changesof 0.1 M ammonium acetate buffer.

Preparation of Anti-Apomucin Antibody. A rabbit polyclonal anti-serum was prepared against the apomucin by immunizing a male NewZealand White rabbit with multiple s.c. injections in the footpad of 25fig of TFMS-deglycosylated mucin preparations emulsified in incomplete Freund's adjuvant. Injections were performed every 2 wk. Serum

was collected and tested for immunoreactivity with apomucin antigenafter 2 mo.

Immunodot Blotting. The cell line extracts were prepared by trypsin-izing the confluent culture, washing in PBS (0.01 M sodium phosphatebuffer containing 0.85% sodium chloride, pH 7.4), and solubilizing thecells in 1 ml of buffer containing 0.5% Nonidet P-40 (Sigma), 0.01 MTris-HCI, pH 7.6, 1 ml of magnesium chloride, and 0.1 mM phenyl-methylsulfonyl fluoride (Sigma). The suspension was incubated on icefor 30 min, followed by centrifugation at 12,000 rpm for 15 min. Theprotein concentration was determined using the BioRad protein assaykit (11). One hundred Mgof protein from each cell line extract andserial dilutions were immunodot blotted. Immunodot blotting wasperformed according to the procedure described by Johnson et al. (12).

2997




PANCREATIC TUMOR APOMUCIN

Chemical Analyses. Mucin or apomucin samples were hydrolyzed in6 N HCI at 105'C for 20 h in a vacuum. The hydrolysates were dried

under vacuum and derivatized with phenylisothiocyanate according tothe method of Heinrikson and Meredith (13). The phenylthiocarbamoylamino acids were separated with an ISCO Model 2350 high-performance liquid chromatography system (ISCO, Lincoln, NE), using an Iscoreversed phase Cti (4.6 x 250 mm, 5 ^m) column inside a water jacket(Alltech Associates, Deerfield, IL) maintained at 52Â°C.Each sample

was performed twice for averaging the amino acid composition. Aminosugars were characterized with the method of Spiro (14). Briefly,samples were dissolved in 4 N HC1 and then dried under vacuum toremove the HCI, and amino sugars were measured on an amino acidanalyzer using a citrate-borate elution buffer.

Binding of Antibodies to Apomucin and Synthetic Peptides. A 24-amino acid synthetic peptide corresponding to the breast mucin gene's

60-nucleotide tandem repeat sequence (15, 16) and a monoclonalantibody HMFG-2 which reacted with breast mucin peptide (6) werekindly provided by Dr. Joyce Taylor-Papadimitriou (Imperial CancerResearch Fund, London, United Kingdom). DU-PAN-2 is a murinemonoclonal antibody against a pancreatic mucin epitope generated inour laboratory (8). The apomucin (200 ng) and synthetic peptide (400ng) were dried onto the wells of a microtiter plate. After blocking thenonspecific binding with 5% bovine serum albumin, rabbit antiserum(1:50) or HMFG-2 or DU-PAN-2 tissue culture supernatant was addedto the plate for l h at room temperature. Bound antibodies weredetected by adding 400,000 to 500,000 cpm/100 n\ of lodogen-labeled(17) goat anti-mouse immunoglobulin (Southern Biotechnology Associates., Inc.) or Protein A (Sigma) as secondary ligand.

Northern Analyses. Total cellular RNA from human pancreatic carcinoma cell lines (HPAF, SW979, Panc-1, Hs766T, Colo 357, T3M4,Aspc-1, and Capan-1), three breast carcinoma cell lines (BT-20, MCF-7, and SKBR-3), and two colon carcinoma cell lines (LS-180 and RW7213) were isolated by the guanidine isothiocyanate/cesium chloridemethod (18). The purified RNA (20 /jg) was analyzed by electrophoresisin 1.2% agarose/formaldehyde gels; this was followed by transfer tonitrocellulose paper. A 60-nucleotide synthetic probe derived from thebreast mucin gene was end labeled with [12P]ATP (Amersham) by the

polynucleotide kinase method (19). The hybridization was performedat 50Â°Cwith 40% formamide, 2x standard saline citrate. 10 Mg/ml ofsheared salmon sperm DNA, and 6x Denhardt's solution.

RESULTS AND DISCUSSION

Pancreatic tumor mucins were partially purified by ammonium sulfate fractionation of HPAF spent culture medium.This method not only concentrated the sample but removed alarge amount of culture medium proteins. The precipitatedsample was further subjected to Sepharose CL-4B molecularsieve chromatography and density gradient ultracentrifugation.This established purification procedure (9) takes advantage ofmucin's high molecular weight being excluded from molecular

sieve chromatography. Since some nonmucin proteins tendedto associate with the mucin molecules, the excluded samplefractions were further purified by CsCl/4 M guanidine HCIdensity gradient centrifugation. The fractions which demonstrated a density (approximately 1.45 g/ml) characteristic ofmucin, together with an absence of contaminating proteins (asassessed by silver staining of 7.5% SDS-PAGE gels of thefractions), were pooled. These samples were subjected to aminoacid analyses which showed that serine, threonine, proline,glycine, and alanine were the major amino acid constituentsand demonstrated an amino acid composition characteristic ofmucin-type glycoproteins (Table 1). The amino acid composition derived from mucin purified from spent supernatant ofHPAF cells reported here was not identical to that reportedpreviously (9) for mucin purified from a patient's ascites fluid,

even though both preparations show compositions consistentwith that expected for mucin. These differences may derive

Table 1 Amino acid composition of pancreatic mucin and apomucin from ahuman pancreatic adenocarcinoma cell line (HPAF)

AminoacidAspartic

acidThreonineSerineGlutamic

acidProlineGlycine/ManineValineMethionineIsoleucineLeucineTyrosinePhenylalanineLysineHistidineArginineGlcNAcGalNAcMucin(residues/

1000residues)45143MX471531481304.ÃŽ191.135NI)91129271296257Apomucin

(TFMS treated)(residues/ 1000

residues)391151567112112511945171945152219.1933N

D"3

" ND, nondetectable (less than 3 residues/1000 residues of amino acid).

from the condition of the two distinct antigen sources (patient's

ascites versus HPAF tissue culture supernatant) in that thepresence of different proteases and glycosidases in these samples may have differentially fragmented the mucins prior totheir biochemical purification, thus causing the relative loss ofdifferent portions of the molecule during their purification. Inaddition, the DU-PAN-2 immunoaffmity chromatography stepused in the previous paper was not used in these studies. Thisprocedure may have removed other populations of mucin molecules bearing no DU-PAN-2 epitope.

Other laboratories have isolated similar mucin-type glycoproteins from tumor cell lines derived from different organ sitesincluding breast and colon (2-5). These have all demonstratedmicroheterogeneity and a characteristic mucin-like amino acidcomposition. Although some blood group determinants havebeen shown to be shared in mucins from different sources, it isclear that these mucin molecules have other carbohydrate determinants which do not cross-react with each other (2, 20).Curiously, there also are apparently significant differences insize and degree of glycosylation among these mucin-type glycoproteins. It is important to determine whether or not thepolypeptide core of these mucin-type glycoproteins shares structural similarity.

In order to strip the carbohydrate side chains, pancreaticapomucin was prepared by TFMS treatment of purified pancreatic tumor mucin. We have reported that this method caneffectively remove over 95% of carbohydrate and retain about70% of proteins (21). Data presented in Table 1 show thecontent of hexosamine to total amino acids as residues/1000amino acid residues of pancreatic tumor mucin. After deglyco-sylation, amino sugar analyses of the TFMS-treated apomucinsrevealed no detectable GlcNAc and only trace amounts ofGalNAc. Five amino acids (serine, threonine, proline, glycine,and alanine) still constituted more than 60% of the amino acidcomposition following TFMS treatment, indicating that thisprocedure caused no significant loss of protein. It is well knownthat GalNAc residues linked to Ser/Thr are resistant to TFMStreatment (7, 22). Lectin binding data indicated that some ofthe GlcNAc and GalNAc residues were still attached to thepeptide core after TFMS treatment as monitored by helixpomatia agglutinin (HPA) and wheat germ agglutinin lectins(data not shown). Nevertheless, after removing most of thecarbohydrate as determined by hexosamine analysis, it was

2998




PANCREATIC TUMOR APOMUC1N

likely that some peptide epitopes were exposed.A rabbit monospecific polyclonal antibody was generated to

TFMS-treated pancreatic apomucin. Deglycosylated pancreaticapomucin and intact pancreatic tumor mucin were immuno-blotted with this rabbit antiserum (Fig. 1). The antiserum failedto react with intact pancreatic tumor mucins (Fig. IB) butreacted with a diffuse M, > 200,000 band in the purifiedapomucin preparation (Fig. \A) which was used to immunizethe rabbit. The molecular size of this TFMS-treated apomucinprobably does not directly reflect the actual molecular size ofthe pancreatic mucin polypeptide core, since at least 5% of thetotal carbohydrate units remained on the TFMS-treated apomucin, thus altering its mobility in SDS-PAGE.

An immunodot blot study was performed by testing extractsfrom various mucin-producing and nonproducing cell lines forreactivity with rabbit polyclonal antibody raised against pancreatic apomucin antigen. As shown in Fig. 2A, the rabbitantiserum reacted with four of five pancreatic (HPAF, SW979,BXPC-3, and T3M4) and one of two breast (BT-20) cell lineextracts but did not react with a colon carcinoma (LS-180) andtwo melanoma (DM-6, SKMEL) cell line extracts. These datasuggested that there were intracellular forms of mucin precursorpresent in pancreatic and breast cell line extracts which sharedsome epitope similarity. These precursor molecules were notseen in a mucin-producing colon cell line extract as well as twomelanoma cell line extracts.

The rabbit antiserum raised against TFMS-treated pancreatictumor mucin was also tested for reactivity with pancreatictumor mucin, pancreatic tumor apomucin, and a synthetic 24-amino acid peptide based on the published sequence for the 20-amino acid breast mucin tandem repeat structure (16) (Fig. 3Ã„).The binding data shown in Table 2 indicated that this rabbitantiserum reacted very strongly with pancreatic apomucin,moderately with breast mucin 24-amino acid synthetic peptide,and showed no reactivity with intact pancreatic tumor mucin.

A B

A murine monoclonal antibody, HMFG-2, reactive with a

breast apomucin epitope (6, 16), demonstrated the same reactivity pattern. The HMFG-2 antibody, which has also beenshown to react with the fully glycosylated breast mucin (6),failed to react with the intact pancreatic mucin but reacted with

B

1

2

3

4

5

6

7

8

9

10

Fig. 2. Immunodot blot. Various tumor cell line extracts (/. HPAF: 2, T3M4;3. SW979: 4. Panc-I; 5. BxPC-3; 6. LS-180: 7. DM-6; 8, SKMEL: 9, SKBR-3;10, BT-20) (100 Mg of protein) were immunodot blotted with rabbit antiserum(1:50) raised against TFMS-deglycosylated pancreatic tumor apomucin (A) orpreimmune rabbit serum (B). The signals were detected by '"I-radiolabeled

Protein A.

MMHI-IK BRI \M Ml (IN I \M)I â€¢M Kl l'I \ I

A. Nucleotide Sequence (Anti-Sense)

Kd

200*-

116*-

94*-

68*-

CGC CCA GGT CAC ACC CTG CGC TGG GGG CGC CGT CCA GCC CGC CGC COG CCT

GOT GTC CGC

B. Peptide Sequence

Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val

Thr Ser Ala Pro Asp Thr Arg

Fig. 3. Synthetic breast mucin tandem repeat. A, 60-nucleotide sequence; B.24-amino acid peptide sequence.

Table 2 Immunohinding assay of antibodies with synthetic breast mucin peptideand TF\fS-treaÃ®edpancreatic tumor mucin

The 24-amino acid synthetic peptide was derived from the breast mucin tandemrepeat sequence (16) as shown in Fig. 3B.

PolyclonalantibodiesPreRS

RSCMonoclonal

antibodiesHMFG-2

DU-PAN-2Breast

mucinpeptide222

Â±98Â°'*

2.010 Â±1122.359 Â±108

142 Â±25TFMS-treated

apomucin260

Â±16315,607 Â±3,3223. 126 Â±832

85 Â±19Pancreatic

mucin127Â±

17132Â± 11160 Â±28

4,143 Â±313

Fig. 1. Western blot. TFMS-treated pancreatic apomucin (A) and purifiedpancreatic tumor mucin (B) were analyzed by 3 to 10% SDS-PAGE. transferredto nitrocellulose, and then immunoblotted with rabbit antiserum raised againstTFMS-treated pancreatic apomucin antigen. The signals were detected by '"I-

radiolabeled Protein A. Preimmune rabbit antiserum was used as negative control.

2999

Â°Mean Â±SD.* Direct binding assay (cpm) of rabbit antiserum detected with l:'I-Protein A

or mouse monoclonal antibodies detected with goat anti-mouse immunoglobulinlabeled with I2!I by the lodogen method (17). See "Materials and Methods" fordetails. Isotype controls for HMFG-2 and DU-PAN-2 were less than 200 cpm inall of the above assays.

1 Rabbit antiserum was generated by immunization with TFMS-deglycosylated

pancreatic tumor apomucin.




PANCREATIC TUMOR APOMUC1N

TFMS-treated pancreatic apomucin. However, an antibody,DU-PAN-2, which reacts with the intact pancreatic mucin (9)was not reactive with TFMS-treated pancreatic apomucin orthe breast mucin synthetic peptide (Table 2). Since it would beimpossible to quantitatively compare binding of mouse monoclonal antibodies and rabbit polyclonal antibodies in this assay,no attempt was made to quantitate the binding reactivities ofthese antibodies. However, the antibody binding data presentedin Table 2 clearly indicated that the rabbit antiserum raisedagainst pancreatic apomucin was cross-reactive with breastmucin synthetic peptide (24-amino acid tandem repeat sequence) as shown in Fig. 3B. TFMS-treated pancreatic apomucin was shown to express the epitope recognized by a monoclonal antibody, HMFG-2, which recognized a breast mucinpeptide epitope (16).

Cross-reactivity of pancreatic and breast mucins has seldombeen observed since the carbohydrate composition and antigenicproperties (9, 23) as well as the tissue distribution (24, 25) ofthese two families of molecules were different. However, arecent report (26) of the establishment of a cytotoxic T-cell linefrom pancreatic cancer patients demonstrated that these T-cell-mediated immune responses recognized a large and heavilyglycosylated mucin molecule expressed on pancreatic and breasttumors and tumor cell lines. Although the nature of the recognized epitope was not determined, it is interesting to speculatethat the common tandem repeat sequence may play a role instimulating the specific cytotoxic T-cell.

In order to further confirm the finding that pancreatic mucinand breast mucin shared some similarity in the tandem repeatsequence, we synthesized a 60-nucleotide probe correspondingto the published tandem repeat sequence (15, 16) as shown inFig. 3A and hybridized this to Northern blots of total cellularRNA (20 ng) that was prepared from 13 different humancarcinoma cell lines. A major 4.4-kilobase message was detected

O. V) I- <

7.46*-

4.40Â»-

2.37*-

1.35*-

Fig. 4. Northern blot. Total cellular RNA (20 ng) from human tumor celllines was electrophoresed in a 1% agarose/formaldehyde gel. transferred tonitrocellulose, and hybridized with a 32P end-labeled 60 mer tandem repeat

sequence probe derived from the breast mucin gene.

3000

in six pancreatic (HPAF, SW979, Colo 357, T3M4, Aspc-1,and Capan-1) and three breast (BT-20, MCF-7, and SKBR-3)carcinoma cell lines (Fig. 4). A significant polymorphism ofmRNA was noticed in the Colo 357 cell line, and similar mRNAsizes were also present above 4.4 kilobases in other cell lines.Two pancreatic cell lines (Panc-1 and Hs766T) as well as twocolon carcinoma cell lines (LS-180 and RW 7213) were completely negative. These results were confirmed with polyade-nylate-containing RNA preparations (data not shown). Finally,we have recently used the rabbit antiserum described above toclone the mucin cDNA from an HPAF X-gtl 1 cDNA library.Sequencing studies on a number of these clones have revealedthe existence of tandem repeat sequences identical to thosereported for the breast mucin gene in this cDNA. We have alsofound these tandem repeat sequences in clones isolated from aX-gtlO cDNA library constructed from the T3M4 pancreaticadenocarcinoma cell line (generously provided to us by Dr.Murry Korc, University of Arizona). These studies will bepublished in a separate report.

Recent studies of a cloned human intestinal/colonie mucingene revealed that a second totally different mucin tandemrepeat sequence existed (27). This 69-nucleotide tandem repeatwas part of a much larger mRNA (7 kilobases) than the breastmucin. In our hands, this 69-nucleotide oligonucleotide probehybridizes to a 7-kilobase mRNA from LS-180, but does nothybridize to HPAF mRNA (data not shown). The existence ofat least two types of mucin genes suggested that differentialglycosylation of breast mucin and intestinal mucin may bedictated by the different polypeptide sequences. However, theantibody binding data and Northern blot analyses in this reportclearly indicated that the tandem repeat protein sequence fromthe breast mucin and a similar mRNA were detected in bothpancreatic and breast mucin-producing cell lines. Comparingthe biochemical data from pancreatic and breast mucins (9, 22),it is difficult to reconcile the glycosylation differences betweenthese two mucin-type glycoproteins. Since the tandem repeatsequence reported to date represents about 30% of the totalgene [comparing 1.4 kilobase of sequence with a mRNA size of4.5 kilobases (16)], one possibility is that, in addition to thetandem repeat sequence, each type of mucin molecule mightpossess its own unique sequence which influences the antige-nicity and the degree of glycosylation of the molecule. Thisquestion and others regarding the differential expression ofmucin molecules in different organ sites will be resolved bysequencing the full length genes for these molecules.

ACKNOWLEDGMENTS

We thank Dr. J. Dawson for helpful advice and critical reading ofthe manuscript. We also thank F. Tuck for technical help, A. Khorramifor performing hexosamine analyses, and Dr. R. R. Randall for hiscollaboration in synthesizing the oligonucleotide probe.

REFERENCES

1. Rittenhouse. H. G., Manderino. G. L.. and Hass. G. M. Mucin-type glycoproteins as tumor markers. Lab. Med., 16: 556-560. 1985.

2. Johnson, V. G., Schlom, J., Paterson, A. J.. Bennett. J.. Magnani. J. L., andColcher. D. Analysis of a human tumor-associated glycoprotein (TAG-72)identified by monoclonal antibody B 72.3. Cancer Res., 46: 850-857. 1986.

3. Lan, M. S., Finn, O. J., Fernsten, P. D., and Metzgar. R. S. Isolation andproperties of a human pancreatic adenocarcinoma-associated antigen. DU-PAN-2. Cancer Res.. 45: 305-310. 1985.

4. Magnani. J. L., Nilsson, B.. Brockhaus. M.. Zopf. D.. Steplewski, Z.,Koprowski. H., and Ginsburg, V. A monoclonal antibody-defined antigenassociated with gastrointestinal cancer is a ganglioside containing sialylatedlacto-A'-fucopentaose II. J. Biol. Chem.. 257.- 14365-14369. 1982.

5. Miotti, S.. Aguanno. S., Canevari, S.. Diotti, A., Orlandi. R.. Sonnino. S..




PANCREATIC TUMOR APOMUCIN

and Colnaghi, M.I. Biochemical analysis of human ovarian cancer-associatedantigens defined by murine monoclonal antibodies. Cancer Res.. 45: 826-832, 1985.

ft. Burchell. J.. Gendler. S., Taylor-Papadimitriou. J.. Girling. A.. Lewis. A..Millis. R., and Lamport. D. Development and characterization of breastcancer reactive monoclonal antibodies directed to the core protein of thehuman milk mucin. Cancer Res.. 47: 5476-5482. 1987.

7. Edge. A. S. B.. Faltynek. C. R., Hof. L., Reichert, L. E.. Jr.. and Weber. P.Deglycosylation of glycoproteins by trifluoromethane sulfonic acid. Anal.Biochem., IIS: 131, 1981.

8. Metzgar, R. S., Gaillard. M. T., Levine. S. J.. Tuck. F. L.. Bossen, E. H.,and Borowitz, M. J. Antigens of human pancreatic adenocarcinoma cellsdefined by murine monoclonal antibodies. Cancer Res., 42: 601-608, 1982.

9. Lan, M. S., Khorrami, A.. Kaufman. B.. and Metzgar. R. S. Molecularcharacterization of a mucin-type antigen associated with human pancreaticcancer. J. Biol. Chem.. 262: 12863-12870. 1987.

10. Carlstedt. !.. Lindgren. H.. Sheehan. J. K.. Ulmsten, U.. and Wingerup. L.Isolation and characterization of human cervical-mucus glycoproteins.Biochem. J., 211: 13-22. 1983.

11. Bradford, M. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. Anal.Biochem., 72; 248-254. 1976.

12. Johnson, D. A., Gautsch. J. W., Sportsman. J. R.. and Elder, J. H. Improvedtechnique utilizing nonfat dry milk for analysis of proteins and nucleic acidstransferred to nitrocellulose. Gene Anal. Tech., /: 3-8, 1984.

13. Heinrikson, R. L.. and Meredith. S. C. Amino acid analysis by reverse-phasehigh performance liquid chromatography: precolumn derivatization withphenylisothiocyanate. Anal. Biochem.. /.J6:65-74, 1984.

14. Spiro. R. G. The carbohydrates of glycoproteins. Methods Enzymol.. 28:11-14, 1972.

15. Siddiqui, J.. Abe. M., Hayes, D.. Shani. Y., Yunis, E.. and Kufe. D. Isolationand sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc. Nati. Acad. Sci. USA. 85: 2320-2323, 1988.

16. Gendler, S.. Taylor-Papadimitriou. J.. Duhig. T.. Rothbard. J.. and Burchell.J. A highly immunogenic region of a human polymorphic epithelial mucin

expressed by carcinomas is made up of tandem repeats. J. Biol. Chem.. 263:12820-12823. 1988.

17. Markwell. M. A. K. A new solid-state reagent to iodinate proteins. Anal.Biochem.. 125: 427-432, 1982.

18. Davis, L. G.. Dibner. M. D.. and Battey, J. F. Basic Methods in MolecularBiology, pp. 130-135. New York: Elsevier. 1986.

19. Davis, L. G.. Dibner. M. D.. and Battey, J. F. Basic Methods in MolecularBiology, pp. 72-74. New York: Elsevier. 1986.

20. Lan. M. S.. Bast. R. C., Jr.. Colnaghi. M. L. Knapp. R. C.. Colcher, D..Schlom. J.. and Metzgar. R. S. Co-expression of human cancer-associatedepitopes on mucin molecules. Int. J. Cancer. 39:68-72, 1987.

21. Lan. M. S., and Metzgar. R. S. Molecular studies of a pancreatic tumormucin, DU-PAN-2. In: UCLA Symposium on Molecular and Cellular Biology, Human Tumor Antigens and Specific Tumor Therapy. Vol. 99. pp. 1-11. New York: Alan R. Liss. Inc.. 1989.

22. Marianne, I., Perini, J. M.. Houvenaghel, M. C., Tramu. G.. Lamblin, G.,and Roussel. P. Action of trifluoromethane sulfonic acid on highly glycosy-lated regions of human bronchial mucins. Carbohydrate Res.. 151: 7-19.1986.

23. Shimizu. M.. and Yamauchi, K. Isolation and characterization of mucin-likeglycoproteins in human milk fat globule membranes. J. Biochem., 91: 515-519, 1982.

24. Borowitz. M. J., Tuck, F. L.. Sindelar. W. F.. Fernsten. P. D., and Metzgar,R. S. Monoclonal antibodies against human pancreatic adenocarcinoma:distribution of DU-PAN-2 antigen on glandular cpithelht and adenocarci-nomas. J. Nati. Cancer Inst.. 72: 999-1005. 1984.

25. Taylor-Papadimitriou. J., Millis, R.. Burchell. J.. Nash, R., Pang. L., andGilbert. J. Patterns of reaction of monoclonal antibodies HMFG-1 and -2with benign breast tissues and breast carcinomas. J. Exp. Pathol.. 2: 247-260, 1986.

26. Barnd. D. L.. Lan, M. S.. Metzgar, R. S., and Finn, O. J. Specific, MHC-unrestricted recognition of tumor-associated mucins by human cytotoxic Tcells. Proc. Nati. Acad. Sci. USA, 86: 7159-7163, 1989.

27. Gum. J. R.. Byrd. J. C.. Hicks, J. W., Toribara. N. W., Lamport. D. T. A.,and Kim. Y. S. Molecular cloning of human intestinal mucin cDNAs. J.Biol. Chem.. 264: 6480-6487. 1989.

3001




polypeptide core of a human pancreatic tumor mucin antigen

Documents