Biochemical Characterization of the Soluble Form of Tumor Antigen MUC1 Isolated from Sera and Ascites Fluid of Breast and Pancreatic Cancer Patients

Vol. 7, 781s-787s, March 2001 (Suppl.) Clinical Cancer Research 781s

Pamela Beatty, Franz-Georg Hanisch, MUC 1 has a heavily O-glycosylated central domain, consisting Donna Beer Stolz, Olivera J. Finn, and of conserved and tandemly repeated stretches of 20 amino acids Pawel Ciborowski z (2). Each tandem repeat has five potential sites of O-glycosylation, which can be substituted with polylactosamine-type chains Department of Molecular Genetics and Biochemistry [P. B., O. J. F., as found on the lactation-associated glycoform of MUC1 (2). In P. C.] and Center for Biologic Imaging [D. B-S.], School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, tumor cells that have originated from ductal epithelia, MUC1 is and Institute of Biochemistry II, University of Cologne, 50931 expressed on the entire cell surface. More importantly, O-linked Cologne, Germany [F-G. H.] oligosaccharides are dramatically reduced in chain lengths, making this form tumor specific and immunologically distinct (3-5). As such, MUC1 is a potential target for immunotherapy Abstract of carcinomas (6). One form of MUC1, a synthetic peptide Transmembrane glycoprotein tumor antigen MUC1 corresponding to five tandem repeats, is already being tested in that is overexpressed on pancreatic and breast tumor cells clinical trials as a vaccine for pancreatic and breast cancers (7). can be found in large amounts in soluble form in serum and Tumor cells release soluble MUC1 (Fig. 1) by an unknown ascites fluid. MUC1 has been identified as a target of human mechanism. This soluble form can be found in large quantities antitumor antibody and CTL responses that are generated in sera and ascites fluid of cancer patients (8). Because of its in the absence of helper T cells. The soluble form of MUC1 abundance, this form is expected to be taken up, processed, and should support generation of helper T cells, but we have presented by APCs 3 to helper T cells. However, this does not found recently that this form, although effectively endocy- appear to be the case, because no MUC 1 helper T cell responses tosed by dendritic cells, remains trapped in early endosomes have been found in cancer patients. Our recent studies showed and is not trafficked to antigen-processing compartments. that MUC1 purified from ascites fluid (ASC-MUC1) is effec- The exact biochemical structure of this form of MUC1 has tively taken up by dendritic cells but then retained indefinitely in not been elucidated to date, and it is thus not clear what early endosomes instead of being processed in antigen-process- structural characteristics may be responsible for its reten- ing compartments (9). We consider this the primary reason why tion in early endosomes. We have purified soluble MUCI patients with so much circulating soluble MUC1 do not generate from ascites fluid of breast/pancreatic cancer patients (ASC- effective immune responses against MUC1 + tumor. Biochem- MUC1) and quantitated O-linked carbohydrates. We have ical properties of this MUC1 form are not known; therefore, we altered ASC-MUC1 by enzymatic treatment: trypsin or clos- undertook detailed characterization of ASC-MUC1 to under- tripain digestion, desialylation, and further in vitro glyco- stand what elements of its structure may influence its processing sylation. We have found that desialylated ASC-MUC1 was and presentation to the immune system. further glycosylated by peptidyl N-acetylgalactosamine The tumor-specific, underglycosylated integral membrane transferases and was not when sialic acid was present. These form of MUC1 is differentiated from the normal, heavily O- alterations created new forms of ASC-MUC1 that might be glycosylated form by specific monoclonal antibodies directed to handled more efficiently by antigen-presenting cells to gen- peptide epitopes within the tandem repeat (10). Monoclonal erate better tumor-specific immunity and used to identify antibodies recognizing tumor-specific MUC1 react with their structures that are directly involved in retention of this epitopes only when O-linked oligosaccharides are substantially antigen in early endosomes. reduced, whereas other mAbs recognize MUC 1, regardless of its glycosylation (11, 12). Our initial characterization of ASC- Introduction MUCl was based on reactivity with 56 anti-MUC1 monoclonal MUC 1, a member of the mucin family, is a large, rod-like, antibodies submitted to the ISOBM TD-4 International Work- transmembrane glycoprotein expressed in a polarized fashion on shop on Monoclonal Antibodies against MUC1. The results the apical surface of ductal epithelial cells (1). In normal cells, from that study showed that ASC-MUC 1 was not recognized by mAbs that react only with underglycosylated (tumor-specific) MUC1 (13), suggesting that the tandem repeat region was glycosylated extensively. However, we hypothesized that the 1 Supported by National Cancer Institute/NIH Grant RO1 CA56103 (to O. J. F.) and a fellowship (to P. C.) from the Alexander von Humboldt Foundation. 2 To whom requests for reprints should be addressed, at Department of Molecular Genetics and Biochemistry, School of Medicine, Biomedical 3 The abbreviations used are: APC, antigen-presenting cell; GalNAc, Science Tower, Room E 1240, University of Pittsburgh, Pittsburgh, PA N-acetylgalactosamine; HPLC, high-performance liquid chromatogra- 15261. Phone: (412) 648-7607; Fax: (412) 383-8859. phy; GlcNAc, N-acetylglucosamine; NeuAc, neuraminic acid.

-Arg-Ser- ~ -Arg-Ala- ~ 85 a'a'-- Tandem repeat regi~ ~ IIIlfflllllll @

-Lys-Asn- PGSTAPPAHGVTSAPDTRPA 1 repeat = 20 a.a. Fig. 1 Schematic representation of ASC-MUCI molecule. Vertical arrows, trypsin and/or clostripain (other than Arg-Pro bonds) cleavage sites that are outside the tandem repeat region (18). Horizontal arrows, the distance from potential cleavage site to the tandem repeat region. *, potential site of N-glycosylation (18).

presence of oligosaccharides may be the cause of inefficient with 50 mg sodium acetate (pH 6.0), and flow-through fractions processing of this form of MUC1. Because glycosylation could containing unbound mucin and other proteins were collected. not be determined from those experiments, we have now under- This preparation was subjected to equilibrium density-gradient taken further analysis and characterization of ASC-MUC1, in- centrifugation in a CsC1 gradient as published previously (15). cluding carbohydrate and protein modifications. Quantitative The final step in the purification was HPLC molecular sieving analysis of O-linked carbohydrates on a monosaccharide level on a BioSep SEC-4000 column (Phenomenex, Torrance, CA). showed that ASC-MUC1 contains 5% w/w of O-linked oligo- MUCl-containing fractions were pooled and extensively dia- saccharides, indicating a substantially reduced degree of glyco- lyzed against distilled H20. The purity of the fractions was sylation compared with forms retained on the surface of normal monitored by SDS-PAGE using 8% gels stained with Coomas- or tumor cells. To characterize individual domains of this sol- sie Brilliant Blue R-250 and immunoblotting. uble form, we altered ASC-MUC1 by sequential enzymatic Electron Microscopy-Molecular Replicas. Lyophilized treatment. Trypsin and clostripain digestion generated fragments ASC-MUC1 was solubilized in 0.5 M ammonium acetate, di- consisting primarily of the tandem repeat region with short luted l:l in glycerol, and then sprayed onto freshly cleaved segments of NH2- and COOH-terminal domains. Neuraminidase mica. Mica was then transferred to a Cressington freeze-fracture treatment removed c~2,3- and oL2,6-1inked sialic acid residues, machine and outgassed to 1 • 10 -7 Torr at room temperature which enabled us to further O-glycosylate this form in vitro by for 5 h. The sample was shadowed while rotating in a plane adding GalNAc residues onto available serine and threonine oriented at a 5 ~ angle with a platinum vapor stream for 12 s at residues. We have generated several new forms of ASC-MUC 1: 80 mA. Samples were overcoated with carbon at a 90 ~ angle for (a) with truncated NH2- and COOH-terminal domains, thus 12 s at 100 mA. Replicas were floated onto double-distilled H20 primarily consisting of the tandem repeat region; (b) enzymat- and then collected onto 200-mesh copper grids. Micrographs ically desialylated; and (c) desialylated and further in vitro were obtained on a JEOL JEM 1210 transmission electron O-glycosylated with radiolabeled [3H]GalNAc as a marker. microscope (Peabody, MA) at 80 kV. SDS-PAGE and Immunoblots. SDS-PAGE was per- Materials and Methods formed as described by Laemmli (16) under nonreducing con- Antibodies. Anti-MUC1 monoclonal antibodies used in ditions, using the Mini-Protean III (Bio-Rad Laboratories, Inc., this study were VU-3C6 and BCP7. Antibody VU-3C6 was Hercules, CA) electrophoresis system. Usually, polyacrylamide obtained from the culture supernatant of a hybridoma gener- gels were 8%. Typical transfer onto nitrocellulose membrane ously provided by Dr. Jo Hilgers (Department of Obstetrics and was overnight at 30 V in Towbin buffer with methanol reduced Gynecology, Academisch Ziekenhuis, Vrije Universiteit, Am- to 10%. A mixture of anti-MUC1 monoclonal antibodies VU- sterdam, the Netherlands) and grown in our laboratory. BCP7 3C6 and BCP7 at 1:1 ratio was used for immunodetection. antibody was from Dr. Ian McKenzie (Austin Research Insti- Monosaccharide Analysis. Monosaccharide analysis tute, Medical Center, Heidelberg, Germany). Epitopes for VU- was performed on ASC-MUC1, which was quantitated by sam- 3C6 and BCP7 are PDTRPAP and HGVTS, respectively (Ref. ple weight after drying overnight in dessicator under vacuum in 12; see Fig. 1 for amino acid sequence of the tandem repeat). the presence of KOH and P205. Known amounts of MUC1 ASC-MUC1 Purification. ASC-MUC1 was purified ac- preparations were resuspended in distilled H20 to a concentra- cording to a protocol published previously (14). Briefly, cell- tion of 1 mg/ml and stored frozen. For any given saccharide free ascites fluid collected from patients with breast or pancre- analysis, aliquots of ASC-MUCI preparations were placed into atic tumors was adjusted to pH 4.7 with 2 M acetic acid and glass tubes and dried again in a dessicator under vacuum in the stirred overnight in the cold. Precipitated proteins were removed presence of KOH and P205. by centrifugation, and the clear supernatant was collected and ASC-MUC1 preparations were hydrolyzed with 2 M triflu- adjusted to pH 6.0 and concentrated 3-fold with Amicon Ultra- oroacetic acid at 121~ for 2 h. The sample was dried under filtration Cell (Amicon, Inc., Beverly, MA) using a PM30 stream of nitrogen with addition of tert-butanol and subjected to membrane with a M r 30,000 cutoff for proteins. Concentrated reduction in the presence of 1 M ammonium hydroxide contain- preparation was run through a CM-Sephadex G-25 (Pharmacia ing sodium borohydride (10 mg/ml) at 4~ overnight. After the Amersham Biotech AB, Uppsala, Sweden) column equilibrated reduction step, borohydride was removed, and alditols were

O-acetylated by the addition of acetic acid anhydride and pyr- buffer (0.25 M sucrose, 1 naN EDTA) at 4~ for 10 min. The idine in a 1:1 ratio and heated for 20 min at 121~ The sample lysate was centrifuged for 20 min at 1000 • g, and the super- was dried under a stream of nitrogen and dissolved in chloro- natant was mixed with 2.5 ml of 0.1 M imidazole buffer (pH form. Acetylated alditols of monosaccharides were analyzed by 7.2), supplemented with 10 naM MnC12 and 200 mM NaC1. After gas chromatography-electroionization mass spectrometry anal- 10 min of stirring, the whole mixture was centrifuged for 20 min ysis (MD800; Fisons, Manchester, United Kingdom) on 15-m at 5000 • g. The pellet was collected and resuspended in 4 ml DB5 capillary column heated from 50~ to 250~ (7.5~ of imidazole buffer with 10 Ixl of Triton X-100. The lysate was The alditol acetates were registered by monitoring the total ion cleared by 1-h ultracentrifugation (120,000 • g), and superna- current and single ions at m/z 145, 217, 289, and 361 (hexose tant containing GalNAc transferases was concentrated with a and pentose) or at m/z 144, 216, 288, and 360 (N-acetylhex- Centristat (Millipore, Milford, MA). osamine). The reaction mixture consisted of 50 txg of a substrate, 5 Ixl O-Linked Glycans Profiling. Oligosaccharide profiles of [3H]UDP-GalNAc (7.8 Ci/mol) as a cosubstrate, and 25 txl of were analyzed after hydrazinolysis of salt-free, dry mucin sam- the preparation of peptidyl GalNAc transferases isolated from ples. Glycans were liberated in hydrazine (Glyco, Novato, CA) MDA-MB-231 cells. The reaction was carried out for 2 h at for 5 h at 60~ dried, and re-N-acetylated according to instruc- 37~ and terminated with 20 mM tetraborate buffer (pH 9.2) tions from Glyko. The reducing glycans were labeled by reduc- containing 1 mM EDTA. After the reaction was terminated, rive amination using a mixture of 2-aminobenzamide (1 M in samples were passed through small columns filled with 0.5 ml acetic acid) and sodium cyanoborohydride (2 M in DMSO), 2:3 of Dowex 1 X 8 (Fluka Chemie AG Buchs, Switzerland) to v/v, and incubation at 60~ for 2 h. The labeled glycans were remove unincorporated [3H]UDP-GalNAc. One fifth of the elu- separated from excess of fluorescent dye by paper chromatog- ant was mixed with 3 ml of scintillation fluid (EcoLite+)l; ICN raphy and finally extracted into 500 Ixl of water. 2-AB-labeled Biomedicals, Inc., Irvine, CA), and incorporated radioactivity oligosaccharides were chromatographed on normal-phase was measured in a 1600TR Liquid Scintillation Analyzer (Pack- HPLC on polymer-based amino-phase column (astec; 0.45 • ard Instruments Co., Meriden, CT). 250 mm) using a gradient of 250 mM aqueous formiate in acetonitrile from 20% to 60% over a period of 90 min. Eluting oligosaccharides were registered by on-line detection with a Results fluorescence detector RF-10A-xl (Schimadzu, Kyoto, Japan) at Rod-like Structure and Heterogeneity of ASC-MUC1 excitation 330 nm (excitation) and 420 nm (emission). The Visualized by Electron Microscopy. A unique feature of the glycans were identified via external standards, which had been MUCl molecule, schematically depicted in Fig. l, is the rod- confirmed by matrix-assisted laser desorption ionization mass like structure of the tandem repeat region, which keeps this spectrometry, and partial desialylation with 0.1 M trifluoroacetic transmembrane molecule extended from the cell surface in vivo. acid treatment (1 h at 80~ prior to rechromatography was Electron micrographs of MUC1 previously, also reported for performed to identify NeuAc-containing oligosaccharides. DuPAN-2 (8), showed elongated, rod-like as well as globular, Trypsin Digestion. ASC-MUC1 preparations were dis- "relaxed" structures of this glycoprotein. Electron micrographs solved in 8 M urea and extensively dialyzed against 10 mM of a sample of ASC-MUC1 purified by us consistently showed Tris-HCl buffer (pH 8.8). Two ixl of trypsin (Promega Corp., MUC1 molecules in a stretched, rod-like form (Fig. 2) of Madison, WI) were added per 120 txg of ASC-MUC1 prepara- different lengths, 680 nm (Fig. 2A), 400 nm long (Fig. 2, B-E), tion. The mixture was incubated for 24 h at 37~ The digest and 280 nm long (Fig. 2F). The size heterogeneity is most likely was subjected to Western blot analysis after separation on attributable to the fact that this preparation is a mixture of SDS-PAGE. ascites from multiple patients, having molecules that differ in Clostripain Digestion. Prior to proteolytic digestion, the number of repeats in the tandem repeat region. clostripain was activated for 3 h at room temperature in the Analysis of the Carbohydrates O-Linked to ASC- presence of 5 mM DTT and 0.5 mg calcium acetate. ASC- MUC1. Quantitative analysis of monosaccharides and sialic MUC1 was resuspended in 10 txl of 25 mM sodium phosphate acid (Table 1) showed that the content of hexoses and hex- buffer (pH 7.5), containing 5 mM DTT and 0.2 mM calcium osamines in ASC-MUC1 was significantly lower than on nor- acetate, and mixed with activated clostripain. Reaction mixture mal MUC1. It was also much lower than on other forms of was incubated for 24 h at 37~ Digestion was terminated by MUC1, such as that isolated from tumor cells grown in culture adding 10 mM EDTA, and the digest was subjected to Western or from tumor tissue (4). The molar ratio of GlcNAc:GalNAc in blot analysis after separation on SDS-PAGE. ASC-MUC1 was 0.23, indicating a very low level of extended Neuraminidase Treatment. Neuraminidase from Clos- oligosaccharides. The w/w ratio of sialic acid:GalNAc in ASC- tridium perfringens was purchased from Calbiochem-Novobio- MUC1 was 1.05, which is lower than the highly sialylated (1.76) chem Corp. (La Jolla, CA). Three hundred txg of ASC-MUC1 and higher than poorly sialylated (0.7) MUC1 forms isolated preparation were treated with 50 milliunits of neuraminidase in from T47D breast carcinoma cells (4). The presence of fucose in 100 mM phosphate buffer (pH 6.0) at room temperature for 4 h ASC-MUC1 makes this form similar to one isolated from in a total volume of 55 pA. MDA-MB-231 cells (4). The average level of glycosylation of In Vitro Glycosylation. Peptidyl GalNAc transferases ASC-MUC1 per tandem repeat cannot be calculated at this time were isolated from breast tumor cells according to the protocol because the average number of repeats in this heterogeneous published by Stadie et al. (17). Briefly, 0.5 ml of a pellet of preparation isolated from several patients is unknown. MDA-MB-231 cells was resuspended in 2.5 ml of extraction Low monosaccharide contents was also reflected in the

Table 1 Monosaccharide composition analysis of glycans on ASC-MUC1 Amounts in Ixg per 100 Ixg of mucin Fuc Man Gal GalNAc GlcNAc NeuAc ASC-MUC1 1.02 1.19 0.984 0.81 0.19 0.85

more slowly. This preparation was digested for 24 h at 37~ and analyzed by Western blot after separation on 8% SDS-PAGE gel. After trypsin cleavage, the two high molecular weight forms disappeared, major species were partially degraded, and two new species with apparent molecular weights of M r 160,000 and M r 110,000, respectively, were generated (Fig. 3, Lane B). Susceptibility to Clostripain Digestion. Clostripain is an Arg-C proteinase that cleaves Arg-Pro bonds that are other- wise resistant to trypsin cleavage (see Fig. 1). However, these bonds become resistant to clostripain cleavage if the tandem repeats are O-glycosylated. A sample of ASC-MUC1 (Fig. 3, Fig. 2 Electron micrograph of ASC-MUC 1 rotary shadowed with plat- Lane A) was subjected to 24-h clostripain digestion at 37~ and inum. MUC1 molecules found in this preparation were 680 nm long (A), analyzed by Western blot after separation on 8% SDS-PAGE 400 nm long (B-E), and 260 nm long (F). gel. Again, the two high molecular weight forms disappeared, and major species were partially degraded, although to a lesser extent than after trypsin digestion. Clostripain-digested ASC- oligosaccharide yields after hydrazinolysis. Normal-phase MUC1 was reanalyzed by separation on 4-15% SDS-PAGE HPLC of fluorescently labeled glycans revealed the presence of gradient gel (Fig. 3, Lane D). Several species with molecular three major sialylated species: NeuAco~2-3Gal[31-3GalNAc weights ranging from M r 150,000 to M r 24,000 were visualized. (-50%), NeuAcc~2-3Gal[31-3(NeuAcoL2-6)GalNAc (-25%), Separation of the clostripain-digested ASC-MUC 1 on a gradient and NeuAco~2-3Gal[31- 4GlcNAc[31 - 6(NeuAcoL2-3Gal[31-3)- gel resulted in further separation of a major species to two GalNAc (--10%; Table 2). Neither neutral nor any fucosylated closely migrating ones. This result indicates that these smaller glycans were detected. An additional NeuAc-containing glycan species represent fragments of larger molecules that were not eluting after the fraction of disialylated glycans was not identi- glycosylated sufficiently to prevent clostripain digestion. fied and found to make up --6% of the total glycans. In sum- Enzymatic Desialylation of ASC-MUC1. ASC-MUC1 mary, the glycosylation profile of ASC-MUCI appears to be was subjected to enzymatic desialylation using a2-3,6-neura- dominated by acidic, sialic acid-containing oligosaccharides. minidase from Clostridium perfringens. This enzyme catalyzes Density estimations of O-glycosylation after deglycosylation/ the hydrolysis of terminal oL2,3 and oL2,6 sialic residues from alkylaminylation in ethylamine and matrix-assisted laser de- complex carbohydrates and glycoproteins. Neuraminidase- sorption ionization mass spectrometry of proteolytic fragments treated and nontreated samples were separated on 8% SDS- revealed that only unsubstituted and disubstituted tandem repeat PAGE gel, and MUC1 was detected by immunoblotting. The peptides were obtained by clostripain cleavage (data not shown). results presented in Fig. 4 show that all neuraminidase-treated Again, this strongly favors the assumption that ascites MUC 1 is species of ASC-MUC1 migrated more slowly in SDS-PAGE O-glycosylated at low density. compared with the nontreated species. Slower electrophoretic Susceptibility to Trypsin Digestion. Each MUC1 tan- mobility of neuraminidase-treated MUC1 is attributable to re- dem repeat contains one arginine-proline bond (see Fig. 1 for the moval of negatively charged sialic acid. A similar effect has sequence), which makes this domain trypsin resistant. Trypsin- been observed previously by us for MUC 1 immunoprecipitated resistant bonds within each tandem repeat and trypsin-suscepti- from neuraminidase-treated cells4 and by others (19, 20). An ble bonds located 76 amino acids NH2-terminal (Lys-Asn) and observed equal shift of all neuraminidase-treated MUC 1 species 54 amino acids COOH-terminal (Arg-Ser) to the tandem repeat suggests that the relative difference in electrophoretic mobility region (18) allow the digestion of ASC-MUC1 with this pro- between these ASC-MUC1 species is attributable to factors teinase to obtain a truncated form primarily consisting of the other than sialic acid contents. tandem repeat region (see Fig. 1). We also expected that such ASC-MUC1 Can Be Further Glycosylated in Vitro Only treatment would remove three of five putative sites for N- in the Absence of Sialic Acid. To determine whether ASC- glycosylation located COOH-terminal to the tandem repeats. MUC1 can be further glycosylated, 100-1xg samples of ASC- ASC-MUC1 preparation subjected to trypsin digestion con- MUC 1 that were untreated, trypsin digested, and enzymatically tained three forms of mucin that are different in their mobility in 8% SDS-PAGE (Fig. 3, Lane A). The major and fastest migrating species shows a molecular weight higher than Mr 185,000 molecular weight standard protein. Two other species migrate 4 J. Magarian-Blander and 0. J. Finn, unpublished data.

Table 2 Profiles of O-linked glycans to the tandem repeats of ASC-MUC1 % of total O-linked Oligosaccharide structure glycans NeuAco~2-6GalNAc-Gal [31-3 Sialyl T (sT) 1.8 NeuAco~2-3Gal[31-3GalNAc- Monosialylated core 1 50.6 NeuAco~2-6NeuAcoL2-3Gal [31-3GalNAc- Disialylated core 1 24.9 [NeuAcc~2-3 ]Gal [31-4GlcNAc [31-6GalNAc- [NeuAco~2-3 ]Gal [31-3 Monosialylated core 2 5.8 a NeuAco~2-3Gal[31-4GlcNAc [31-6GalNAc-NeuAcc~2-3Gal [31-3 Disialylated core 2 10.7 Non-identified, sialic acid-containing glycan 6.2 "The exact position of NeuAc is not known.

A B C D A B

18s = 18s 185 kDa ~.~

4 ~i 84184:;~)~J~i 119 kDa ,~ 119~ 119 kDa 8s 85 kDa ~.~ 62 kDa 85 kDa

51 kDa 62 kDa --.t,,.- 62 kDa

51 kDa 51 kDa

38 kDa ~ 14 38 kDa

Fig. 4 Western blot analysis of neuraminidase-treated ASC-MUCI. Fig. 3 Western blot analysis of proteolytically altered ASC-MUC1. ASC-MUC 1 before (Lane A) and after 4 h treatment with neuraminidase ASC-MUCI before (Lane A), after 24 h trypsin digestion (Lane B), and (Lane B). Samples were separated by SDS-PAGE in 8% gel under alter 24 h clostripain digestion (Lane C). Samples were separated by nonreducing conditions. The membranes were blotted using a mixture of SDS-PAGE in 8% gel under nonreducing conditions. Clostripain di- 3C6 and BCP7 anti-MUC1 monoclonal antibodies in a 1:1 ratio. gested ASC-MUC1 re-analyzed by SDS-PAGE in 4-15% gradient gel under nonreducing conditions (Lane D). The membranes were blotted using a mixture of 3C6 and BCP7 anti-MUCl monoclonal antibodies in 1: 1 ratio. several patients. One important observation is that the density of O-linked oligosaccharides on the tandem repeats of ASC-MUC l is lower than expected. Muller et al. (21) previously reported desialylated, were subjected to in vitro glycosylation using that soluble form of MUC1 isolated from culture supernatant of [3H]UDP-GalNAc as a cosubstrate by a crude preparation of T47D breast cancer cells is glycosylated at high density with an GalNAc transferases isolated from MDA-MB-231 breast tumor average 4.8 GalNAc residues per one repeat and O-linked sac- cells. Concurrently, 50 txg of 100-mer synthetic peptide repre- charide chains are short. This further means that almost all senting five tandem repeats of MUC1 were used as a substrate potential sites of O-glycosylation were substituted, yet the w/w to monitor the effectiveness of glycosylation. Desialylated ASC- ratio of saccharides:protein is low in this form. The authors MUCI was glycosylated at a higher rate than nonglycoslated postulate that high density O-glycosylation of MUC 1 with short synthetic peptide, whereas forms carrying sialic acid were not oligosaccharide chains is a characteristic of other tumor cell further glycosylated (Fig. 5). Two conclusions can be drawn lines. 5 In contrary, normal MUC1, which by w/w ratio of from this result: (a) ASC-MUC1 contains multiple nonglyco- saccharides to protein is considered highly glycosylated, has sylated sites, --1.5 times more than nonglycosylated peptide substituted only half of potential sites of glycosylation and representing five tandem repeats; and (b) the presence of already O-linked oligosaccharides are long and branched. Our prepara- sialylated oligosaccharides blocks the transfer of GalNAc resi- tion of MUC 1 contained only nonglycosylated or diglycosylated dues onto available serine and threonine residues. tandem repeats (data not shown). One possible explanation of this fact is that tumor cells in vivo produce much higher levels Discussion We report in this study the results of quantitative carbohydrate analysis of ASC-MUC1 and its further enzymatic modi- 5 S. Miiller and F-G. Hanisch, Recombinant MUC1 VNTR fusion pro- fications on the carbohydrate and protein levels. The preparation tein expressed in human breast cancer cells reveals strongly fluctuating used in our experiments represents a native, soluble, form that individual profiles of mainly core 2-based O-linked glycans, submitted was derived from a mixture of ascites fluids obtained from for publication.

2500 that frequency of sequence variants in the amino acid sequence of the polypeptide tandem repeat region can be as high as 50%. 2O00 We conclude from our data, however, that substitution of Pro residues located COOH-terminally to Arg residue is much less 1500 frequent than other reported errors. In addition to the major M r 200,000 species, clostripain digestion generated several other 1000 fragments with smaller molecular weight. These smaller fragments were generated by the cleavage of Arg-Pro bonds within the tandem repeat region, which were not protected by O- 500 glycosylation. Neuraminidase treatment of ASC-MUC1 removed a2,3 0 and oL2,6 sialic residues, reflected by a slower electrophoretic mobility (less negative charge) in SDS-PAGE. The neuramini- o ~ ~ ~ dase-treated form of ASC-MUC1 could be successfully further .r glycosylated by peptidyl GalNAc transferases isolated from Fig. 5 In vitro glycosylation of ASC-MUC1 using GalNAc trans- MDA-MB-231 breast tumor cells, whereas forms that still carry ferases isolated from MDA-MB-231 breast tumor cells and [3H]GalNAc sialic acid could not. This result indicates that ASC-MUCI has as a cosubstrate. Three forms of ASC-MUC 1 were used as a substrate: multiple serine and threonine residues that have not been gly- desialylated with neuraminidase (ASC-Ne); trypsin digested (ASC-Tr); cosylated with GalNAc. Thus, the presence of sialic acid on and nontreated (ASC-MUC1). Synthetic MUCI, a 100-mer peptide, was concurrently used as a positive control. Glycosylation is expressed as a glycosylated serines and threonines prevents even the first step number of cpm representing incorporated [3H]GalNAc. of glycosylation of other serine and threonine residues. This observation is in agreement with relatively low w/w ratio of saccharides:glycoprotein in this preparation. In summary, we have found that the soluble form of MUC 1 of sialyltransferases than those in in vitro culture, which is in serum and/or ascites fluid is glycosylated at lower levels than reflected by the low density O-glycosylation and short oligo- suggested previously, but this amount of glycosylation still saccharide chains that are highly sialylated. This agrees with our prevents efficient processing of this antigen by APCs. Further- observation discussed below, that the presence of sialic acid on more, our in vitro glycosylation experiments showed that ASC- the already O-linked glycans interferes with initial step of gly- MUC 1 has serine/threonine residues still available for the initial cosylation of the neighboring sites. step of O-glycosylation, transfer of GalNAc residues, which can Quantitative analysis of monosaccharides showed that occur only in the absence of sialic acid on already glycosylated ASC-MUC1 contains fucose residues, although we have not residues. Enzymatic modifications have generated additional found fucosylated oligosaccharide species in our preparation. forms of soluble MUC1 that might be taken up and processed There are two possible explanations of this discrepancy. Either more efficiently by APCs. This will allow us to study the the level of oligosaccharide species containing fucose was under positive and negative effects of more extensive glycosylation of detection threshold of HPLC detector, or fucose detected in the this form on antigen processing. monosaccharide analysis originated from N-linked glycans. To further modify ASC-MUC1, we took advantage of several already known structural features of the MUC1 glyco- References protein: (a) the repeats have an Arg-Pro bond that is trypsin 1. Gendler, S. J., and Spicer, A. P. Epithelial cell mucins. Ann. Rev. resistant, whereas the NH 2- and COOH-terminal domains have Physiol., 57: 607-634, 1995. trypsin-susceptible cleavage sites. Trypsin digestion also re- 2. Hanisch, F-G., Uhlenbruck, G., Peter-Katalinic, J., Egge, H., Dab- moves three of five potential sites of N-glycosylation (Fig. 1); rowski, J., and Dabrowski, U. Structures of neutral O-linked polylac- (b) this Arg-Pro bond, although normally susceptible to clostri- tosaminglycans on human skim milk mucins. J. Biol. Chem., 264: pain digestion, becomes resistant when the tandem repeat of 872-883, 1989. MUC1 is glycosylated beyond Tn antigen (GalNAc-Ser/Thr). 3. Lloyd, K. O., Burchell, J., Kudryshov, V., Yin, B. W. T., and Taylor-Papadimitriou, J. Comparison of O-linked carbohydrate chains Trypsin digestion generated three MUC1 species with mo- in MUC-1 mucin from normal breast epithelial cell lines and breast lecular weights around M r 200,000, M r 160,000, and M r carcinoma lines. J. Biol. Chem., 271: 33325-33334, 1996. 100,000, respectively. The M r 200,000 species was also gener- 4. Hanisch, F-G., Stadie, T. R. E., Deutzman, F., and Peter-Katalinic, J. ated by clostripain digestion, and in both cases, species migrat- MUC1 glycoforms in breast cancer. Cell line T47D as a model for ing slower in SDS-PAGE disappeared. We conclude that this M r carcinoma-associated alterations of O-glycosylation. Eur. J. Biochem., 200,000 species is a truncated form containing O-glycosylated 236: 318-327, 1996. tandem repeat regions. Observed smaller fragments generated 5. Brockhausen, I., Yang, J. M., Burchell, J., Whitehouse, C., and by trypsin but not by clostripain digestion are most likely Taylor-Papadimitriou, J. Mechanisms underlying aberrant glycosylation of MUC 1 mucin in breast cancer cells. Eur. J. Biochem., 233:607-617, attributable to amino acid replacement in the tandem repeats. 1995. One such replacement introducing a new trypsin-susceptible 6. Barrat Boyes, S. M. Making the most of mucin: a novel target for bond, His-Gly to Val-Arg, has been found in the tandem repeats tumor immunotherapy. Cancer Immunol. Immunother., 43: 142-151, of MUC1 isolated from MCF-7 cells. Muller et al. (21) reported 1996.

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Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2001 American Association for Cancer Research. Biochemical Characterization of the Soluble Form of Tumor Antigen MUC1 Isolated from Sera and Ascites Fluid of Breast and Pancreatic Cancer Patients

Pamela Beatty, Franz-Georg Hanisch, Donna Beer Stolz, et al.

Clin Cancer Res 2001;7:781s-787s.

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