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

(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.Â¡,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 ÃŸ-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 ÃŸ-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>

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 Â¿Â¿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%). We chose to use a high ionic strength serine and threonine level for muc-\ is less than or equal to 5()c/rof the buffer to reduce the electrostatic interactions between molecules. A pH of 5.9 level found in muc-3 and muc-4. In addition, muc-\ contains 20% was chosen for the D2O studies in Figs. 2, 3, and 4 to avoid perturbations of glycine, and muc-3 and muc-4 do not contain any glycine. These the spectra resulting from the partial protonalion of histidine, but significantly differences in amino acid content between muc-\ and muc-3 and different from the pKa value of histidine. The I-dimensional 'H NMR experi muc-4 is reflected in the numbers of reverse turns per repeat predicted ments in H2O were performed at pH 6.8 for Fig. 5. The tree amino acids in by the Chou and Pasman criteria (Table 1). The muc-l and muc-2 Figs. 3 and 4 were at the same stoichiometry as found in the muc-1 tandem genes are similar in proline content, yet differ greatly in threonine, repeat. A Bruker AM-5(K) NMR spectrometer equipped with Aspect 3000 computer serine and threonine, and glycine content. There are twice as many and a 5-mm 'H probe was used to record the spectra of the mucin muc-1 turns predicted for 100-amino acids of muc-\ than for muc-2. It is peptides. The spectra were recorded at 25Â°C,with the temperature of the probe currently believed that mucin peptide backbones are random coil and regulated with a BVT-1000 unit and calibrated with a mcthanol sample. The ultimately derive their structure from glycosylation. Our hypothesis is D2O spectra of the peptides were recorded 5 to 10 min after dissolution. that the repetitive region of muc-l exists as a polyproline ÃŸ-turnhelix Suppression of the water signal was accomplished during the repetition delay and that this form of secondary structure is compatible with extensive of 1.5 s for peptide samples in D2O and H2O. The one-dimensional spectra glycosylation. were recorded following a single 90Â°Cpulse. A control spectrum of the H2O Characterization of Tandem Repeat Peptides. The sequence of sample was taken without water presaturation to ensure that none of the amide the muc-1 TR domain is (PDTRPAPGSTAPPAHGVTSA)â€ž. Each re protons were affected by presaturation of water signal at any given power level. peat starts with a proline in position 1 and ends with alanine in A total of 1024 transients were collected for each spectrum. The two-dimen position 20. To examine the protein core structure of this 20-amino sional correlated spectrum was recorded in a phase-sensitive mode. A sine hell acid repetitive domain, we prepared 20-, 40-, and 60-amino acid long filter was applied to the time domain data in both Fl and F2. The acquired data synthetic peptides corresponding to one-, two-, and three-tandem re size was 2048 x 1024 points. Zero filling was used to obtained a final data matrix of 4096 x 4096 points. All proton chemical shifts were relative to the peats. The analytical HPLC profiles (Fig. 1, top) of the unpuritied mucin muc-1 synthetic peptide products and electrospray mass spec reference compound DSS at 0.0 ppm. Circular Dichroism. The circular dichroism spectra were recorded on a trum (Fig. 1. bottom) of the major peptide component resolved by Japan Spectroscopic Company (Jasco) Model J-710 circular dichroism spec- HPLC (shown by arrow) are shown in Fig. 1. The striking fidelity of tropolarimeter (Hachioji City, Japan). The temperature was controlled by using synthesis and resultant purity as shown in the HPLC profiles are a Jasco PTC-343 Peltier-type thcrmostatic cell holder and temperature control believed to be a result of the unusual structure and sequence of muc-1. program. The spectrum was recorded from 180-260 or 195-260 nm with In each case the peptide molecular weight determined by mass spec- readings every 0.1 nm at 25, 55, 75, and 90Â°C.The peptide concentration was troscopy was the expected molecular weight calculated from amino 0.1 mg/ml of HPLC-purified peptide in 0.01 Mphosphate buffer at pH 7.2. A acid content. The peaks on the mass spectrum represent massxharge 0.1-cm path length strain-free quartz cuvet was used to record the spectrum. ratios with each tandem repeat capable of acquiring 2 additional The solvent spectrum was subtracted from that of mucin and a noise reduction charges and hence 2 peaks. subroutine was applied to the resultant spectrum. A total of 10 scans were Monoclonal Antibodies to Native muc-1 Recognize Synthetic accumulated for each sample. No change in the solvent spectrum was observed Peptides. To verify that the synthetic peptides corresponding to one-, with increasing temperature. two-, and three-tandem repeats of muc-1 protein core fold into the Intrinsic Viscosity. All viscometry measurements were performed by using a Cannon-Fenske-Ostwald type capillary viscometer with HPLC-purified pep native structure, the peptides were reacted with a panel of muc-1 - tide in O.I M phosphate buffer at pH 7.0 and 30Â°C.The procedure was as specific monoclonal antibodies (Table 2). These antibodies were pre described previously (19. 20). The capillary constant was calculated as re viously shown to react with epitopes specific for the carcinoma- ported by Tanford and Buzzell (19). The kinematic viscosity measurements associated form of muc-1 (10, 17). The antibodies were reacted were repeated at least 10 times, and the averages were used to calculate the against equal quantities of the synthetic peptides in a solid-phase intrinsic viscosity. Intrinsic viscosity was calculated from kinematic viscosity, and the appropriate density correction (0.0029 ml/g) was applied as recom Table 1 Sequence Â«/m/v.v/.vofhuman tnucin luntletn repeats mended (21). The Simha shape factor and the peptide axial ratios were calcu Mucin gene lated according to Refs. 22 and 23. Molecular Modeling of the 60-Amino Acid Peptide. The sequence of the TR domain of the human mucin mÂ»c-l(24) gene was modeled into a poly-type No.acids/repeatr-f of amino ofproline% I turn conformation on a silicon graphics model INDIGO (Mountain View. CA) ofIhreonine% terminal, using the Tripos molecular graphics program Sybyl (St. Louis. MO). ofglycine<7r Using this model the longitudinal axis and cross-sectional axis were measured, ThrNo.of Ser + and the axial ratio (longitudinal/cross-sectional) of the 60-amino acid peptide of turns/repeat"muc-120251520253muc-22421624622muc-3176410701muc-4166250501 was estimated. ' Predicted by Chou and Pasman rules. 5387

20 mer (1 repeat) 40 mer (2 repeats) 60 mer (3 repeats)

2.0-ECo(MC\JÂ§1.0-1oconJ 2.0- 2.0

1.0- 1.0-

Fig. 1. The analytical HPLC chromalograms of the crude synthetic peptide products of the 20-, 4Ãœ-. and 60-amino acid peptides are shown above. The 15 20 25 30 35 40 15 20 25 30 35 40 15 20 25 30 35 40 elcclrospray mass spectra of the largest HPLC peak % Acetonitrile % Acetonitrile % Acetonitrile fractions (indicated by arrow) are shown below. In each case the mass obtained was the expected mo lecular weight (20 mer = 18X6, 40 mer = 3756. 60 1126 MW mer = 5625). 11-10 MW 1252 MW 1886liti 3756 30 5625 â€¢¿9 7 - 940 I â€¢¿ 25- 6 - 8-XH 1406 5 - 20-

4 - 15 -J <Â§5-Co4 3 - -3 10- -2 2- -1 5- 1 - -buia]944 lodali i^Li^Xu,j|g

700 1000 1300 1600 700 1000 1300 1600 700 1000 1300 1600 Mass/Charge Mass/Charge Mass/Charge

enzyme-linked immunosorbent assay. The reactivity is defined as the (28, 29). The increase in reactivity of the monoclonal antibodies with slope of the color change with time. the 40- and 60-amino acid peptides indicates that the epitopes attain a Most antibodies failed to react with 20-amino acid peptide corre native conformation in the absence of glycosylation, reflecting the sponding to one repeat and beginning with proline 1 (Table 2). How structure seen in native mucin. ever, these antibodies reacted with peptides corresponding to two- and Mucin Protein Core Forms a Stable Folded Secondary Struc three-tandem repeats of the protein core. A probable explanation for ture. Two-dimensional correlated spectroscopy by 'H-Proton NMR this is that native presentation of the predominant epitope (PDTRP) in DiO can reveal amide protons (N:::H) that typically are involved in recognized by these antibodies requires at least the alanine of the hydrogen bonding critical to the formation of secondary structure. The previous repeat. This observation could explain the results obtained by observation of cross-peaks between amide protons and a in deuterium others showing that other amino acids can be substituted for alanine. indicates that these protons are protected from exchange with the and that peptides linked to a carrier or a pin will react without alanine solvent. A fingerprint region of the two-dimensional correlated spec- 5388

Table 2 Monwlonal antibody recognition of nnicin peptitie* Reactivity in ELISA"

AntibodyHMFG2139H2175C5201E9BC1BC2BC3EpitopcDTRCore?i(A)PDTR(A)PDTR(A)PDTR20mer4.52.2Ã•3297.02.74.840mer173013UHI271710060mer173436121141883

" ELISA, enzyme-linked immunosorbent assay.

troscopy of the mucin 60-amino acid peptide in D2O (Fig. 2) clearly shows cross-peaks of some nonexchangeable amide protons. This particular region of the spectrum shows scalar correlation between amide-'H and 'H-a protons. These cross-peaks did not exchange during the duration of more than 12 h at 27Â°C.Thus, these amide protons appear to be protected very well inside the 3-dimensional structure of the folded mucin. This experiment clearly shows that a mucin 60-amino acid peptide retains a stable ordered structure in solution, in distinct contrast to the random coil conformation previ ously reported (15). Development of Structure Requires Multiple Tandem Repeats. Fig. 3 shows the region of the 'H NMR spectrum which is charac teristic of ÃŸ-protonsof the amino acid side chains. The muc-1 tandem repeat sequence contains only one aspartic acid (D) and one histidine (H) residue per TR, and the side chain ÃŸ-protonsof these amino acids are resolved into two distinct regions of the spectrum (30). Fig. 3 shows the spectrum of the free amino acids as compared to the spectrum of the synthetic peptide corresponding to one-tandem repeat, two-tandem repeat, and three-tandem repeat peptides. Arrows indicate differences in the spectra associated with increasing numbers of tan ppm dem repeats in the peptide. These spectra indicate that the develop Fig. 3. 'H NMR spectra of mucin peptides dissolved in deutcrated 0.1 M phosphate ment of an ordered structure depends on the number of tandem repeats buffer. pH 5.89. in D2Ã›.showing the region of the ÃŸ-protonsof aspartic acid and histidine. (size) in the peptide. If the secondary structure of these peptides were Development of structure depends on the number of tandem repeats in the peptide. random coil, the spectrum in this region would be expected to be

independent of the number of repeats present and to correspond closely to that of the free amino acids (30). The data in Fig. 3 show clearly that the spectrum is dependent on the number of repeats and is significantly different from the spectra observed for free amino acids. Free amino acids, or peptides containing one, two, or three 20- amino acid repeats of muc-1 core all contain the same information when considering the 'H NMR responsive protons in the region of the spectrum from 1.6 to 3.3 ppm from DSS (30). Differing chemical shifts and numbers of peaks are the result of changes in the local magnetic fields arising from structural changes (folding) of the pep tide backbone. Of particular interest in Fig. 3 are the distinct spectral changes occurring in the aspartic acid ÃŸ-protonresonances (2.4 to 2.7 ppm) when going from free amino acids to one-, two-, and three- tandem repeats. Similarly, structural changes are evident from the changes in the histidine ÃŸ-protonresonances (2.9 to 3.3 ppm) as the number of protein tandem repeats increases. These results can be interpreted to indicate that an ordered structure is not completely formed in a peptide with only one 20-amino acid repeat, and that the larger peptides containing 2 and 3 tandem repeats contain sufficient 5.5 5.0 4.5 1.0 3.5 folding information to result in a cooperative formation of structure. IH (ppm ) Insight into the equivalence of particular residues within each copy Fig. 2. Five hundred-Mil/ correlation spectroscopy 'H NMR spectra of muc-1 60- of the tandem repeat was obtained from the side chain protons of amino acid pcptide dissolved in deulerated 0.1 M phosphate buffer, pH 7.2. in D;O. The histidine when dissolved in D2O (Fig. 4). The amino acid histidine cross-peaks show scalar correlation between amide-'H and amide-'Ha. These amide protons are protected from exchange with the solvent by the folded structure of the mucin contains two nonequivalent side chain protons known as C2 (8.3 ppm) peptide. and C4 (7.1 ppm) (Fig. 4). The chemical shifts of the C2 and C4 5389

to three repeats or 60-amino acids (results not shown). The spectral pH 5.89 changes seen between 3.6 and 3.9 ppm support the hypothesis that the muc-1 protein core develops an ordered secondary structure as the number of repeats increases above one. 60 mer Circular Dichroism Spectrum Is Characteristic of Proline-rich (3 repeats) Proteins. The CD spectrum of the 60-amino acid synthetic peptide (3 repeats) contains a large negative peak at 198 nm (Fig. 6). This spectrum is characteristic of proline-rich repeat proteins known to form nonglobular extended structures, including bovine elastin, C hordein, collagen, polyproline II, and wheat gluten (31-33). This spectrum is identical to that observed with the model compound 40 mer A'-acetyl-L-proline-Nv/V-dimethylamide with proline in the trans con- (2 repeats)

20 mer (1 repeat)

Free amino acids

8.5 8.0 7.5 7.0 ppm Fig. 4. 'H NMR spectra of mucin peptidcs dissolved in dcutcralcd 0.1 M phosphate buffer, pH 5.8Â«.in D:O, showing the region of the C2 (8.2-8.4 ppm) and C4 (7.1-7.2 ppm) side ch;iin ring protons of histidine. A single peak for each type of proton in peptides containing multiple histidines indicates that the environments of the histidincs are equiva lent.

protons are very sensitive to local environment. They are always â€”¿Tâ€” resolved from each other by distinct magnetic resonances and are 8.5 8.0 7.5 7.0 ppm usually separated by about 1 ppm. The resonance of the C2 proton in the free amino acid (Fig. 4a) is significantly shifted from the C2 Fig. 5. 'H NMR spectra of mucin peptides dissolved in H2O and O.I M phosphate resonance of the 20-amino acid peptide (Fig. 4i>). We found that the buffer, pH 6.8. (a) Twenly-amino acid peptide corresponding to one tandem repeat, (ft) Forty-amino acid peptide corresponding to two tandem repeats, (c) Sixty-amino acid two histidines in the 40-amino acid peptide which had the potential for peptide corresponding to three tandem repeats. 4 distinct resonances in this region (6.5 to 8.5 ppm) were resolved into only two peaks (Fig. 4c). The same was true with the three histidines in the 60-amino acid peptide which had the potential for 6 distinct resonances and were resolved into only 2 very sharp peaks (Fig. 4rf) in D,O. Finding only one His C2 and one His C4 resonance for a peptide containing 2 or 3 histidines indicates that each histidine is in exactly the same conformation and local environment. The region 'H NMR spectrum which is characteristic of amide protons [(N::H) 6.5 to 8.5 ppm] is shown in Fig. 5. When synthetic peptides corresponding to 1, 2, and 3 copies of the muc-1 repeat are dissolved in 0. l Mphosphate buffer, pH 6.8 in H2O, numerous changes are evident throughout the region indicating changing H-bonding patterns with increasing number of protein repeats. The region of the 'H NMR spectrum from 3.6 to 3.9 ppm contains the resonances of the 8-protons of proline (results not shown). The 'H NMR spectra in 210 220 230 240 250 260 deuteratcd 0.1 M phosphate buffer, pH 5.89 in 99.9% D2O reveals Wavelength (nm) numerous subtle changes in the proton resonances throughout this Fig. 6. Circular dichroism spectrum of mucin 60-amino acid peptide in 0.1)1 M phos region as the number of repeats increases from one, to two, and finally phate buffer. pH 7.2, at 25, 55, 75, and 90Â°C. 5390

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. SECONDARY STRUCTURE OF MUCIN mut-] PROTEIN CORE formation in which the large negative Cotton effect at 198 nm is peptide assumes an ordered conformation in solution, in agreement attributed to three IT-TV*transitions and a large n-Tr* transition in the with the structure suggested by previous NMR experiments. tertiary amide (34, 35). In contrast, the model compound (/V-acetyl- Intrinsic viscosity can also yield information about molecular t,-proline-iV,A'-dimethylamide) with proline in the civ conformation shape. The intrinsic viscosity for all globular proteins is 3.3 to 3.9 ml/g yields a CD spectrum with a large positive peak at 198 nm. The and is independent of molecular weight (22). The value of 7.71 ml/g resultant spectrum is the linear combination of the spectrum of the tor the muc-1 peptide with 3 repeats rules out a globular shape and is Irans and cis isomers (31, 34). The spectrum obtained here indicates consistent with a rod-like shape with an axial ratio (length/width) of that the //wi.v-proline chromophore dominates the spectrum of the 9.2 (23). This measured axial ratio value (9.2) is in agreement with the muc-1 peptides. Increasing the temperature would be expected to value of 9.7 determined from the molecular graphics program Sybyl result in a lowering of the energy barrier between trans- and cis- in which the peptide sequence was modeled as series of type I reverse proline, thus resulting in the formation of more c/.v-proline. and de turns. creasing the negative intensity at 198 nm (31, 34). This prediction is We can conclude from the intrinsic viscosity value that the peptide supported by the 23% decrease in intensity at 198 nm as the tempera with 3 repeats forms an ordered conformation in solution that is ture is increased from 25, to 55, 75, and 9()Â°C(Fig. 6). There arc at rod-like in shape with a longitudinal span of 33-34 A/repeat. This least 2 possibilities to explain this reduction in ellipticity with increas result suggests that the unglycosylated protein core could determine ing temperature. One is that the formation of more d.v-proline isomer the extended structure seen in electron micrographs (39). These results also support the hypothesis that the muc-1 protein core exists as a results in a less intense negative CD effect. Another is that the pro polyproline ÃŸ-turnhelix. gressive loss of secondary structure may explain the decreasing CD intensity with temperature. From modeling studies of other proline- Model of a Polyproline /Â¡-Imn Helix for muc-1 TK Domain. Fig. 7 shows a computer model of the 60-amino acid peptide in the rich repeat proteins, proline primarily in the trans conformation is favored to predominate in a polyproline ÃŸ-turnhelix form of second polyproline ÃŸ-turnhelix conformation that was created by assuming that the mucin sequence exists in a poly-type 1turn conformation. This ary structure (16). Thus, the CD results obtained with mucin could be model reveals that the amino acid side chains radiate outward from an explained by a combination of both mechanisms, the formation of extended rod-like backbone, and are completely exposed to the sol c/.v-proline residues and the loss of overall secondary structure in the vent (Fig. 7). This orientation of the side chains facilitates accessibil molecule. ity of potential glycosylation sites to the glycosylation machinery. The Further interpretation of the CD spectrum of the mucin 60 mer showing a large negative peak at 198 nm is that it contains no a-he- secondary structure is not necessarily dependent on glycosylation. nor does it have to be disrupted by the addition of carbohydrate. This lical or ÃŸsheet content and is representative of the random-coil model explains the lack of effect that heating the peptides has on the conformation (36, 37). In order to address the random-coil issue, we NMR spectrum. Since no unfolding can occur in the globular sense have compared the CD spectra of a peptide with no proline (TAE- with side chains moving from a buried hydrophobic core to an aque NAEYLRV), the mucin 20 mer, and the mucin 60 mer in phosphate buffer at 25Â°C.The shapes of the spectra are all similar, but dramatic ous exterior, there are no large chemical shifts of the side chain protons upon heating (28). The model also explains why the A and DT differences were found in intensity at 198 nm. The ratio of the molar residues will permit substitution within the primary epitope of AP- ellipticities (|(-)]mucin 60 mer/[(")] control peptide) is 13.6. In contrast, DTRP. When the turn is formed, the P and R amino acid side chains the ratio of the molar ellipticities ([(-)]mucin 20 mer/[(")| control pep are in the same space and accessible for binding to the antibody. tide) is 3.2. The molar ellipticity of the 20-amino acid peptide is much Substitutions that allow the turn to form will be tolerated. closer to the value obtained for a 10-amino acid peptide with no proline. In contrast, the molar ellipticity of the 60-amino acid peptide shows a very intense CD band relative to the control. The difference DISCUSSION in molar ellipticities between the 20- and 60-amino acid peptides can The use of peptides corresponding to specific domains and frag only arise from the secondary structure in the larger peptide. This result supports our 'H NMR and antibody-binding results showing ments of larger proteins to study and make inferences about the structure of the larger native protein has significant precedent in the that the development of structure requires multiple repeats. While "random coil" peptides may form transient helixes that display the literature (40-45). It has previously been found that peptides rich in proline are often able to assume native-like conformations in solution. same CD spectrum as the mucin 60-amino acid peptide, the relative Some examples of proline-rich fragments of larger proteins that have intensity as measured by the molar ellipticity reveals that these struc been studied are Type I and IV collagen (42, 46), LCI alkali light tures account for more than 10-fold less in the random coil peptide. chain of skeletal myosin (47), bovine elastin (43, 48), and the Spisula Intrinsic Viscosity Measurements Support a Folded Rod- solidissima nuclear sperm-specific protein (49). shaped Structure. The intrinsic viscosity [TJ] ml/g is a sensitive Our two-dimensional 'H-NMR (COSY) experiment in D2O with measure of the state of folding, and the molecular shape (globular the use of a 60-amino acid synthetic peptide shows that the mucin vcrsux rod-like) of a protein (22, 38). Tanford (38) has shown that for tandem repeat domain can fold into a stable structure, and that this a protein in a random-coil state, the intrinsic viscosity [T)| ml/g is at structure is capable of sequestering protons from exchange by deute a maximum and is given by the equation [TJ] ml/g = 0.684 Â«Â°67, rium for more than 24 h. In addition, one-dimensional 'H NMR where n is the number of amino acids in the protein. The random coil experiments in D2O with the synthetic peptide analogue correspond intrinsic viscosity of a protein depends only on the number of resi ing to one-, two-, and three-repeats of the tandem repeat domain, show dues. For a 60-amino acid peptide the intrinsic viscosity value is that there are structural changes occurring with increasing number of predicted to be 10.7 ml/g. The measured value for the muc-1 synthetic repeats. Evidently, the structural changes are occurring throughout the peptide with 3 repeats is 7.71 ml/g. This value of 7.71 ml/g would length of the 20-amino acid repeat domain, as changes can be detected correspond to the expected intrinsic viscosity of a random-coil 36- throughout the molecule by focusing on the ÃŸ-protonsin the region of amino acid peptide. The measured value of intrinsic viscosity for the 2.4 to 3.3 ppm from DSS (Fig. 3). By concentrating on the ÃŸ-protons muc-1 peptide with 3 repeats is significantly less than expected if the we have taken advantage of the peculiar repetitive nature of this peptide were random coil. Therefore, based on intrinsic viscosity, this protein domain. One 20-amino acid peptide contains all of the protons 5391

Fig. 7. The sequence of three-tandem repeats of the human mucin muc-1 gene modeled into a poly-type I turn conformation by using the Tripos molecular graphics program Sybyl.

that can contribute to the 'H NMR spectrum, and any differences peptides came to the conclusion that the CD spectrum, similar to the observed in the spectrum of peptides corresponding to one-, two-, or one we show here, represents that of a random-coil secondary struc three-tandem repeats can only be attributed to changes in the local ture (52). However, the model peptide used to represent random-coil magnetic environment imposed through the development of second secondary structure was (PKLKL),,. This is a proline-rich repetitive ary structure along the polypeptide backbone. Clearly, the 'H NMR sequence peptide which also fits well into the model of a proline-rich spectra reveal that the structures of peptides containing one-, two-, and ÃŸ-turnhelix. Indeed, a turn is predicted to occur every (LPKL) by three-repeats are different. However, peptides containing multiple his- applying the Chou and Pasman criteria for predicting turns to the tidyl residues whose C2 and C4 resonances resolve into single peaks (PKLKL),, repetitive sequence (25). This earlier interpretation should suggests that the environment of each histidine in multiple repeat be reconsidered in light of our data and the accumulating number of peptides are equivalent. Our interpretation of the 'H NMR results is proline-rich repetitive sequence proteins that also show this type of that the precise conformation of a residue depends on the number of CD spectrum. Examples include C hordein (32), collagen and poly- repeats in the peptide. Previous studies on muc-1 core structure, using proline II (31), bovine elastin (33), and nuclear sperm-specific protein an 11-amino acid fragment of muc-1 tandem repeat, were able to show from Spisula solidissima (49). An alternative interpretation is that this that a reverse-turn structure formed when dissolved in dimethyl sulf- is the characteristic CD spectrum of the polyproline ÃŸ-turnhelix oxide, from D2 through P4, and that P4 existed in the trans confor secondary structure with predominantly irans-proline. Dukor and mation (50, 51). In the present study, using much larger synthetic Keiderling (53) have shown that the CD spectrum of random-coil peptides, we have demonstrated that there is a gradation of structures structures is also typified by a negative peak at 198 nm. This is due to that depends on the size of the peptide. the fact that small peptides, once thought to exist in a random-coil This interpretation of the above 'H NMR data is strongly supported conformation are not random at all, but form transient left-handed by the monoclonal antibody-binding data. Many of the monoclonal 3]-helixes like that found in poly-t-proline II, which consists of all antibodies failed to react with a peptide corresponding to just one fraw-proline (53). repeat, even when the epitope was present, but increasing numbers of Matsushima et al. (16) proposed a structural model for proline-rich repeats resulted in increased antibody reactivity. This behavior is tandem repeat domains that is based on four specific criteria. Analysis consistent with that found by other authors which show that providing of our results for the criteria suggested by Matsushima reveals the amino acids COOH-terminal to the first proline forms the major following, (a) Fulfilling the criteria for the amino acid content, the immunodominant epitope (28, 29). muc-1 tandem repeat domain contains 20% proline, high glycine and The 'H NMR experiments clearly show that the mucin tandem serine content, and contains high alanine instead of phenylalanine or repeat domain assumes an ordered structure in solution, and insight tyrosine. (b) Mucin contains a low predicted o helix and ÃŸsheet into the form that the structure may take comes from analysis of the content and a high content of predicted ÃŸturns,(c) Results of circular mucin sequence, the shape of the molecule obtained from intrinsic dichroism studies support the low a helix and ÃŸ-sheetpredictions and viscosity measurements and electron microscopy, and the circular the high ÃŸ-turnpredictions. This includes the circular dichroism spec dichroism studies. A previous study on the CD spectrum of model trum dominated by the irans-proline chromophore, the high molar 5392

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. SECONDARY STRUCTURE OF MUCIN muc-1 PROTEIN CORE cllipticity as compared to a peptide without proline, and the decrease aptophysin, synexin, gliadin. RNApolymerase II, hordein, and gluten. Proteins Struct. in molar ellipticity upon heating, indicating the formation of cis- Funct. Genet., 7: 125-155, 1990. 17. Jerome, K. R., Bu, D., and Finn, O. J. Expression of tumor-associated epitopes on proline or the melting of secondary structure, (d) Viscometry studies Epstein-Barr Virus-immortalized B-cells and Burkilt's lymphomas transfected with indicating a rod-like structure. Therefore, the tandem repeat domain of epithelial mucin complementary DNA. Cancer Res., 52: 5985-5990, 1992. 18. Fonlenol. J. D., Ball, J. M., Miller, M. A., David. C. M., and Montelaro, R. C. A muc-1 satisfies all of the criteria put forth by Matsushima et al. (16) survey of potential problems and quality control in peptide synthesis by the fluo- to form a poly-proline ÃŸ-turnhelix form of secondary structure. renylmethoxycarbonyl procedure. Pept. Res., 4: 19-25, 1991. The demonstration that the protein core of human mucin muc-1 19. Tanford. C.. and Buzzcll. J. G. The viscosity of aqueous solutions of bovine serum albumin between pH 4.3 and 10.5. J. Phys. Chem., 60: 225-231, 1956. protein core folds into a stable ordered structure in solution, and the 20. Buzzcll, J. G., and Tanford. C. The effect of charge and ionic strength on the viscosity extended rod shape and repetitive nature of muc-1 support the expla of ribonuclease. J. Phys. Chem., 60: 1204-1207, 1956. nation previously put forth for the major histocompatibility complex 21. Tanford, C. Intrinsic viscosity and kinematic viscosity. J. Phys. Chem.. 59: 798-799, unrestricted T-cell reactivity against mucin, seen in patients with 1955. 22 Tanford, C. Physical chemistry of macromoleculcs pp. 798-799. New York: John breast and pancreatic adenocarcinomas expressing this protein (8, 14, Wily & Sons, 1961. Cantor. C'. R.. and Schimmel. P. R. Biophysical Chemistry, Part 2: Techniques for the 17). T-Cell receptors specific for the muc-1 core are thought to be 23. Study of Biological Structure and Function. New York: W. H. Freeman & Co.. 1980. activated through cross-linking of the receptors by the highly re 24. Gcndler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., and peated, regularly spaced, T-cell epitopes (54). In addition, knowledge Taylor-Papadimitrou, J. Cloning of partial cDNA encoding differentiation and tumor- associated mucin glycoproteins expressed by human mammary epithelium. Prix;. of the structure of this protein may enhance our ability to design better Nati. Acad. Sci. USA, 84: 6060-6064, 1987. synthetic peptide vaccines which contain precise structural epitopes. 25. Chou. P. Y.. and Fasman, G. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1993 American Association for Cancer Research. Biophysical Characterization of One-, Two-, and Three-Tandem Repeats of Human Mucin (muc-1) Protein Core

J. Darrell Fontenot, Nico Tjandra, Dawen Bu, et al.

Cancer Res 1993;53:5386-5394.

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