Cancer Letters, 55

(1990)

13-

16

13

Elsevier Scientific Publishers Ireland Ltd.

Scanning tunneling microscopy imaging of the tumour associated antigenic 20 amino acid human polymorphic epithelial mucin core peptide fragment M.C. Davies”, D.E. Jacksona, “The VG STM Laboratory for Research Campaign Laboratories, (Received (Accepted

M.R. Priceb and S. J.B. Tendler”

Biological Uniuersify

Applications, Department of Nottingham, Nottingham

of Pharmaceutical NG7 2RD (U.K.)

Sciences

and

Tancer

7 August 1990) 11 Sepfember 1990)

Introduction

Summary Human

polymorphic

(PEM)are

high

epithelial

molecular

mucins

weight glycoproteins

that are associated

with breast cancer. Recent studies haoe identified that the protein core of PEM contains a 20 amino acid tandem repeat that has elements of secondary structure which coincide with the epitopes for a number of tumour reactiue antibodies. In our continuing structural studies we have now

structural

investigated

the use of the scanning

tunneling

(STM) to directly image the conformation of the twenty amino acid PEM core peptide. High resolution STM images reveal

Human polymorphic epithelial mucins (PEM) are complex high mol. wt. glycoproteins that are associated with human breast carcinoma [l]. The protein core of the macromolecule has been shown to consist of a repeating unit of 20 amino acids I that has been shown to be antigenic [2]. Pro-Asp-Thr-Arg-Pro-Ala-Pro-Gly-Ser-ThrAla-Pro-Pro-Ala-His-Gly-Val-Thr-Ser-Ala

(I)

microscope

that

the

similar

ing. The peptide

peptide

has

an ouerall

to that predicted images identify

directly

is conformationally

can adopt a number in the solid state.

topography

by molecular

modell-

that the free

non-restricted

and

of discrete conformations

Keywords: scanning tunneling microscopy; mucin; conformation; antigenic peptides

Correspondence Biological

to: M.C.

Applications,

Davies, The VG STM Laboratory Department

ences, University of Nottingham,

0304-3835/90/$03.50

0

of

Pharmaceutical

Nottingham

for Sci-

NG7 2RD, U.K.

Recent immunological studies have identified that the peptide contains the epitopes for a number of tumour reactive antibodies including the arg-pro-ala-pro epitope for the antibody C595 [3] and the asp-thr-arg epitope for the antibody HMFG-2 [3,4]. Structural studies utilising high field NMR spectroscopy have identified a type-1 p-turn to be present in the region of asp-thr-arg suggesting that this structural motif is involved in antibody recognition of the intact PEM [5]. In order to further these structural and epitope mapping studies we have investigated the use of the scanning tunneling microscope (STM) to directly image the conformation of these antigenic peptides. Over the last few years, STM has emerged as a powerful tool for the direct imaging of the

1990 Elsevier Scientific Publishers ireland Ltd

Published and Printed in Ireland

14

surfaces of a diverse range of materials with near atomic resolution within various environments [6]. High resolution images have been obtained on specific biological materials, including DNA [7] and phospholipid membranes [8]. We have now extended the potential of this technique by obtaining STM images of the tumour associated 20 amino acid PEM core peptide I. Materials and Methods The preparation and purification of peptide I was as previously described [3]. Samples were prepared for the STM by the deposition of 200 ~1 of a 0.018-mM peptide solution in trifluoroethan01 onto a freshly cleaved clean graphite surface. Trifluoroethanol was selected as a solvent as it is known to induce secondary structure in peptides. The adsorbed peptide was examined in air after solvent evaporation without the use of a thin conducting coat. In order to ensure STM resolution, routine images of the graphite substrate were obtained at atomic resolution throughout this study. Images were obtained on a VG Microscopes STM 2000

(East Grinstead, U.K.) in constant current mode with a sample bias of + 20 mV and a tunneling current of 100 pA. Molecular modelling was undertaken on an Evans and Sutherland PS390 molecular modeller using Biosym’s (Basingstoke, U.K.) Insight software. Results and Discussion A 500 x 350 w image of the sample prepared with the peptide is shown in Fig. 1. A number of discrete surface units with similar dimensions are clearly visible, these units we identify to be individual peptide molecules. The peptides appear to cluster in the vicinity of the step ridge of the graphite substrate. While peptides l-3 are seen as discrete units, peptide 5 appears to lay on the side of peptide 4. The cluster 6 appears to consist of a number of peptide units. A higher resolution STM image (220 X 160 A) of peptides 1 and 2 is presented in Figs. 2 and 3 as a normal and Xmodulated image respectively. The surface dimensions for peptide 1 measures 50 x 21 x 10 A, a distinct kink is visible in this peptide. Peptide 2 is in a more compact conforma-

Fig.l. 500 x 350A STM image of peptide [clustered at a step ridge (A-B) of a freshly cleaved graphite surface.

15

Fig. 2.

Higher resolution image (220

x

160 A) of peptides 1 and 2 in Fig. 1.

tion of dimension 27 x 34 x 10 8,. The dimensions of peptides 1 and 2 reflect that the different topography observed is due to the folding of peptide 2. In order to investigate these dimensions, the peptide was graphically constructed in an elongated conformation. The distance between the

Fig. 3.

amino nitrogen of proline (1) and the carboxyl oxygen of alanine (20) was measured to be 53 8,. Although it is difficult to assess the exact conformation of the peptide in the solid state, the similarity between the len h of the model elongated peptide and the 50 x long peptide 1 (Figs. 2 and 3) is striking. Further STM studies

X-modulated representation of Fig. 2, indicating the heights of the peptides and illustrating a kink in peptide 1.

16

and image analysis projects are now under way in this laboratory to relate these conformations observed with data from other biophysical and immunological investigations. These images show directly that the PEM core peptide I can exist in a number of discrete conformations when deposited upon a graphite substrate. Whilst NMR spectroscopy and Xray diffraction studies provide structural data on such systems, these techniques may have problems identifying the subpopulations present in the sample. The STM images of the peptide that we have presented here directly identify the individual conformations of the peptide present in the sample. The ability to directly determine the conformation of such tumour-associated peptides shows the potential of this new technique to contribute to the understanding of the relationship between biological activity and structure. Acknowledgements S.J.B.T. acknowledges the receipt field Foundation New Science Award. We thank Dr M. Cox and Dr of VG Microscopes (East Grinstead, technical assistance.

of a NufLecturers P. Griffin U.K.) for

References Price, M.R. (1988) High molecular weight epithelial mucins as markers in breast cancer. Eur. J. Cancer Clin. Oncol., 24, 1799-1804. Gendler, S., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J. and Burchell, J. (1988) A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J. Biol. Chem., 263,12820-12823. Price M.R., Hudeu, F., O’Sullivan, C., Baldwin, R.W., Edwards, P.M. and Tendler, S.J.B. (1990) Immunological and structural features of the protein core of human polymorphic epithelial mu&. Mol. ImmunoI., in press. Burchell, J., Taylor-Papadimitriou, J., Boshell, M., Gendler, S. and Duhig, T. (1989) A short sequence, within the amino acid tandem repeat of a cancer-associated mucin contains immunodominant epitopes. Int. J. Cancer, 44, 691-696. Tendler, S.J.B. (1990) Elements of secondary structure in a human epithelial mucin core peptide fragment. B&hem. J., 267,733-737. Binnig, G. and Rohrer, H. (1985) The scanning tunneling microscope. Sci. Am., 253, 50-56. Arscott, P.G., Lee, G., Bloomfield, V.A. and Evans, D.F. (1989) Scanning tunneling microscopy of Z-DNA. Nature (London). 119,484-486. Zasadzinski, J.A.N. (1989) Scanning tunneling microscopy with applications to biological surfaces. Biotechniques, 7, 174-187.

Scanning tunneling microscopy imaging of the tumour associated antigenic 20 amino acid human polymorphic epithelial mucin core peptide fragment.

Human polymorphic epithelial mucins (PEM) are high molecular weight glycoproteins that are associated with breast cancer. Recent structural studies ha...
1MB Sizes 0 Downloads 0 Views