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Biophysical Characterization of One-, Two-, and Three-Tandem Repeats of Human Mucin (muc-1) Protein Core¹

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 [N. T., C. H.], Pittsburgh, Pennsylvania 15213

Until recently mucin tandem repeat protein cores were believed to exist in random-coil conformations and to attain structure solely by the addition of carbohydrates to serine and threonine residues. Matsushima et al. (Proteins Struct. Funct. Genet., 7: 125–155, 1990) recently proposed a model of the secondary structure of proline rich tandem repeat proteins that has challenged this idea, especially for the case of the human polymorphic epithelial mucin encoded by the muc-1 gene. We report here results of structural analyses of the muc-1 protein core by using synthetic peptide analogues. Synthetic peptides were prepared to correspond to one-, two-, and three-tandem repeats of muc-1. Results of one- and two-dimensional ¹H NMR correlation spectroscopy on these peptides confirm that the muc-1 protein core is not in a random-coil secondary structure. Long-lived amide protons are protected in D₂O, and increasing spectral complexity in the region of the ß-proteons of Asp 2 and His 15 reveals that structural changes are occurring as the number of repeats increases. The greatest changes occur when the number of repeats increases from one to two.

These results are supported by the reactivity of a panel of monoclonal antibodies raised against tumor associated muc-1 with these synthetic peptides in enzyme-linked immunosorbent assay. The primary immunodominant mucin epitope, PDTRP, does not appear to attain a native conformation in the single repeat peptide (20 amino acids, starting with P), but is expressed on peptides with multiple repeats. Intrinsic viscosity measurements of the peptide containing three repeats indicate that an ordered structure present in solution is rod shaped. The circular dichroism spectrum of the same peptide is dominated by proline in the trans conformation. These results are all consistent with the prediction that the muc-1 tandem repeat polypeptide core forms a polyproline ß-turn helix.

¹ This 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.).

² Present address: Theoretical Biology and Biophysics, Los Alamos National Laboratory, T-10 Mail Stop-K710, Los Alamos, NM 87544.

Received 4/26/93. Accepted 9/ 9/93.

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