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(CANCER RESEARCH 53.5386-5394, November 15. 1993] Biophysical Characterization of One-, Two-, and Three-Tandem Repeats of Human Mucin (muc-1) Protein Core1 J. Darrell Fontenot,2 Nico Tjandra, Dawen Bu, Chien Ho, Ronald C. Montelaro, and Olivera J. Finn Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine /J. D. F., D. B., R. C. M.. O. J. F.], Pittsburgh 15261, and Department of Biological Science, Carnegie Mellon University fN. T., C. H.¡,Pittsburgh, Pennsylvania 15213 ABSTRACT structural understanding of the precise mucin epitopes present on tumors and normal tissues must be acquired. Until recently mm in tandem repeat protein cores were believed to exist Until recently mucin TR-' protein cores were believed to exist in in random-coil conformations and to attain structure solely by the addi random-coil conformations and to attain structure solely by the addi tion of carbohydrates to serine and threonine residues. Matsushima et al. (Proteins Struct. Funct. Genet., 7: 125-155, 1990) recently proposed a tion of carbohydrates to serine and threonine residues (15). However, model of the secondary structure of proline rich tandem repeat proteins newly acquired information on mucin protein sequences through that has challenged this idea, especially for the case of the human poly complementary DNA cloning has challenged this idea, especially for morphic epithelial mucin encoded by the muc-l gene. We report here the case of human polymorphic epithelial mucin encoded by the results of structural analyses of the muc-1 protein core by using synthetic muc-\ gene. The sequence and amino acid content of the repetitive peptide analogues. Synthetic peptides were prepared to correspond to domain of human muc-\ gene are more compatible with the formation one-, two-, and three-tandem repeats of muc-1. Results of one- and two- of a polyproline ß-turnhelix type of secondary structure (16). It is dimensional 'II NMR correlation spectroscopy on these peptides confirm difficult to accept that a sequence which is repeated faithfully up to that the muc-1 protein core is not in a random-coil secondary structure. 200 times would show no structural preference. It is much easier to Long-lived amide protons are protected in Do. and increasing spectral postulate that a core sequence would in fact be conserved and repeated complexity in the region of the ß-protonsof Asp 2 and His 15 reveals that structural changes are occurring as the number of repeats increases. The in part due to its precise structural and physical properties which are greatest changes occur when the number of repeats increases from one to necessary to construct the entire protein. We recently reported the two. unusual recognition of native muc-1 protein core by T-cell antigen These results are supported by the reactivity of a panel of monoclonal receptors and the subsequent activation of the T-cells, which implied antibodies raised against tumor associated muc-1 with these synthetic that the receptor was recognizing a precise 3-dimensional structure (8, peptides in enzyme-linked immunosorbent assay. The primary immuno- 14, 17). In the current study, we have performed structural character dominant mucin epitope. I'D I UP. does not appear to attain a native ization of muc-1 synthetic peptides of 20, 40. and 60 amino acids in conformation in the single repeat peptide (20 amino acids, starting with P), length, corresponding to one-, two-, and three-tandem repeats, by 'H but is expressed on peptides with multiple repeats. Intrinsic viscosity NMR spectroscopy, CD spectroscopy, intrinsic viscosity (TI)measure measurements of the peptide containing three repeats indicate that an ments, and reactivity with monoclonal antibodies. We have examined ordered structure present in solution is nul shaped. The circular dichroism spectrum of the same peptide is dominated by proline in the imti\ con the relationship between the tandem repeats and development of higher order structure in the human muc-1 protein core and have formation. These results are all consistent with the prediction that the muc-1 tandem repeat polypeptide core forms a polyproline /Mm u helix. proposed a model that is compatible with the observed antigenicity of this core structure. INTRODUCTION MATERIALS AND METHODS Mucins characteristically are large secreted and/or transmembrane glycoproteins with greater than 50% of their molecular weight derived Synthesis of Tandem Repeat Peptides. All peptides were peptide amides from O-linked carbohydrate attached to serine and threonine residues and were synthesized by a manual solid-phase strategy by using y-fluorenyl- (for a review see Ref. l). The bulk of the glycosylation is contained methyloxycarbonyl-protected amino acids. The procedures for synthesis, pu within a domain composed of tandemly repeated sequences of 10-81 rification, and characterization of the peptide products are described in detail amino acids per repeat (2-6). Mucins are produced by cells of epi elsewhere (18). Briefly, 20-, 40-, and 60-amino acid peptides were synthesized thelial lineage and, recently, expression of certain epitopes has been independently by using a manual Rapid Multiple Peptide Synthesizer apparatus identified as being associated with tumors (7, 8). Studies with mono from Du Pont (Boston. MA). When a peptide chain reached 30 amino acids in length, the total resin was separated into two reaction cartridges, thus allowing clonal antibodies reactive with epithelial tumors and corresponding sufficient space for the growing peptide chains in the cartridge. Once the resins normal tissues reveal that there can be different epitopes associated were divided, the concentration of input amino acid was maintained at 0.5 rnsi with mucins from malignant cells as opposed to normal cells (8-10). in order to drive the coupling reaction to completion with high efficiency. The This is in part due to aberrant glycosylation in certain tumors which products of the synthesis were deprotected and cleaved from the resin support results in the exposure of the mucin tandem repeat protein core on the in concentrated trifluoroacetic acid in the presence of the appropriate scaven cell surface (7, 9, 11-13). The exposure of the protein core of certain gers. The trifluoroacetic acid-soluble products were extracted sequentially in mucins found on malignant cells, combined with the ability of the organic solvents and then transferred to water and lyophilized. The peptides immune system to respond to these structures (8, 14) offers a unique were purified by conventional gel filtration and reverse-phase HPLC. Molecu opportunity to utilize mucin-based vaccines for specific immuno- lar weight characterizations of the peptide products were performed with therapy of tumors. For this approach to be viable, a more detailed electrospray mass spectroscopy. Solid Phase Peptide Enzyme-linked Immunosorbent Assay. Peptides containing one, two, and three tandem repeats were bound to %-wcll plates by Received 4/26/93; accepted 9/9/93. overnight incubation in 0.05 Mbicarbonate buffer. Next, the remaining protein- The cosls of publication of this article were defrayed in part by the payment of page binding sites were blocked with a 1-h, room temperature incubation in 10% charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grants RO1CA (O. J. F.), RO1CA 43216 (R. C. M.), GM-26874 (C. H.). and National Science Foundation Grant DMB-8816384 (C. H.). 3 The abbreviations used are: TR. tandem repeat: NMR. nuclear magnetic resonance; - Present address: Theoretical Biology and Biophysics. Los Alamos National Labora CD, circular dichroism; HPLC. high-pressure liquid chromatography; PBS. phosphate- tory, T-10 Mail Stop-K710. Los Alamos, NM 87544. buffered saline: DSS. 2.2-dimethyl-2-silapentane-5-sulfonate. 53Xf> Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. SECONDARY STRUCTURE OF MUCÕN muc-1 PROTEIN CORE Carnation non-fat dry milk in PUS at pH 7.4. The plates were then incubated The TR domains of human mucins muc-1. 2, 3, 4 were also modeled with 50 fxl of the appropriately diluted monoclonal antibodies for 1-h at room according to the rules of Chou and Pasman (25) for secondary structure prediction. Surface potential was predicted by using the "SurfaccPlot" algo temperature. The plates were then washed 3 times with PBS. followed by a 1-h incubation with 50 ftl of the secondary antibody consisting of anti-mouse IgG rithm as described (26). Potential amphipathic a-helical regions were predicted by using the "Amphi" algorithm of Margalit et al. (27). The results of these or anti-mouse IgM conjugated to alkaline phosphatase and diluted 1/3000 in 10% Carnation nonfat dry milk in phosphate-buffered saline at pH 7.4. The analyses were used to construct conformational models (results not shown). plates were then washed 3 times with PBS. Detection was accomplished with The number of predicted turns per repeat is summarized in Table 1. 3 mg/ml phosphatase substrate in 0.25 M diethanolamine with 68 ¿¿M MgCI2-6H2O at pH 9.8. The absorbance at 405 nm was read at 5-min intervals. RESULTS The reactivity is the slope of the change in absorbance over time. Nonspecific antibodies yield a slope of 0-2 and so we considered anything with a slope over Sequence Analysis of Human Mucins. Sequence analysis of the 5 to be positive. repetitive sequences of human mucins reveals that fundamental dif 'H NMR Spectroscopy of TR Peptides. 'H NMR analyses were per ferences exist between the peptide sequences predicted by the muc-l formed by using HPLC-purified and lyophilized peptides. The concentrations gene and the genes of muc-2, -3, and -4 (Table 1) (2^1, 6). The proline used were from f>-7.5 mm in 0.1 M phosphate buffer, pH 5.9, with either level is 4 times higher in muc-\ than in muc-3 and muc-4, and the H2O/D2O (90%/10%) or D2O (99.9%).

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