BIOPHYSICAL CHARACTERIZATION OF ONE-TANDEM, 2-TANDEM, AND 3-TANDEM REPEATS OF HUMAN MUCIN (MUC-1) PROTEIN CORE

Until recently mucin tandem repeat protein cores were believed to exis t in random-coil conformations and to attain structure solely by the a ddition of carbohydrates to serine and threonine residues. Matsushima et al. (Proteins Struct. Funct. Genet., 7: 125-155, 1990) recently pro posed a model of the secondary structure of proline rich tandem repeat proteins that has challenged this idea, especially for the case of th e human polymorphic epithelial mucin encoded by the muc-1 gene. We rep ort here results of structural analyses of the muc-1 protein core by u sing synthetic peptide analogues. Synthetic peptides were prepared to correspond to one-, two-, and three-tandem repeats of muc-1. Results o f one- and two-dimensional H-1 NMR correlation spectroscopy on these p eptides confirm that the muc-1 protein core is not in a random-coil se condary structure. Long-lived amide protons are protected in D2O, and increasing spectral complexity in the region of the beta-protons of As p 2 and His 15 reveals that structural changes are occurring as the nu mber of repeats increases. The greatest changes occur when the number of repeats increases from one to two. These results are supported by t he reactivity of a panel of monoclonal antibodies raised against tumor associated muc-1 with these synthetic peptides in enzyme-linked immun osorbent assay. The primary immunodominant mucin epitope, PDTRP, does not appear to attain a native conformation in the single repeat peptid e (20 amino acids, starting with P), but is expressed on peptides with multiple repeats. Intrinsic viscosity measurements of the peptide con taining three repeats indicate that an ordered structure present in so lution is rod shaped. The circular dichroism spectrum of the same pept ide is dominated by proline in the trans conformation. These results a re all consistent with the prediction that the muc-1 tandem repeat pol ypeptide core forms a polyproline beta-turn helix.