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balt3/bc-bc/bc-bc/bc2901/bc3203-01a

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THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

ARTNO: M103187200 Vol. 276, No. ??, Issue of ???? ??, pp. 1–xxx, 2001 Printed in U.S.A.

Identification and Topology of Variant Sequences within Individual Repeat Domains of the Human Epithelial Tumor Mucin MUC1* Received for publication, April 10, 2001, and in revised form, May 11, 2001 Published, JBC Papers in Press, May 11, 2001, DOI 10.1074/jbc.M103187200

Katja Engelmann‡, Stephan E. Baldus§, and Franz-Georg Hanisch‡¶ From the ‡Institute of Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany, and §Institute of Pathology, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 9, 50931 Cologne, Germany

AQ: A

AQ: B

AQ: C

Fn1

ever, in a previous study, we were able to show that MUC1 tandem repeats of a human breast cancer cell line contain a series of sequence variations on the protein level (11). The concerted replacement of Asp2-Thr3 3 Glu-Ser was revealed at high incidence (in the order of 50% of the individual number of repeat units), and the single replacement of Pro13 3 Ala was demonstrated to occur in approximately 30% of the repeats (11, 12). Evidence for variations on the protein level was also reFn2 vealed for MUC1 from individual human milk samples.2 No nucleotide sequence information was reported to confirm these AQ: D data on the DNA level and to exclude that some of the amino acid replacements were the result of posttranslational events, nor were these sporadic findings corroborated as related to the general structural features of human MUC1. The occurrence of substitutions within the icosapeptide of the MUC1-VNTR domain is of high biomedical significance, particularly with regard to the concerted replacement of two adjacent positions in the immunodominant DTR motif. This motif is a preferred epitope in murine B-cell responses (4) and also in humans (2) and represents the primary target site for major histocompatibility complex-unrestricted cytotoxic T-cell responses in breast cancer patients (13). Classical major histocompatibility complex-restricted responses to epitopes within the tandem repeat have been reported for many mouse strains (14), and human cytotoxic T lymphocytes recognizing MUC1 peptide epitopes were also demonstrated (15), including major histocompatibility complex class II-dependent helper T-cell responses in vitro (16). The results of these experimental studies have found application in a series of clinical studies. For example, anti-MUC1 peptide antibodies, like the DTR recognizing HMFG1, have been used to deliver high doses of yttrium to the AQ: E peritoneum of ovarian cancer patients, and these initial trials were recently extended in a Phase III multicenter clinical trial (17). Beyond antibody strategies, active specific immunotherapy based on MUC1 peptides has been performed in syngeneic and transgeneic mouse models, including naked DNA, viral vectors, peptides, and liposome encapsulation of peptides (17). These attempts have led to a series of Phase I clinical studies using tandem repeat peptides of MUC1 in conjugation with a variety of carriers (17). The accumulating evidence from laboratories and clinics makes MUC1 tandem repeat peptide a primary immunotarget in anticancer strategies. Accordingly, structural aspects of the VNTR domain, which were not realized in previous sequencing studies of the gene (7–10), are of utmost importance for the design of efficient tumor vaccines.

This study shows for the first time that the tandemly repeated icosapeptide of human MUC1 underlies a genetic sequence polymorphism at three positions (underlined): PDTRPAPGSTAPPAHGVTSA. The concerted replacement DT3 ES (sequence variation 1) and the single replacements P3 Q (sequence variation 2), P3 A (sequence variation 3), and P3 T (sequence variation 4) were identified by sequencing of polymerase chain reaction products and studied by minisatellite variant repeat analysis for their incidence and topology in the 5ⴕ and 3ⴕ peripheral regions of the variable number of tandem repeats domain. Minisatellite variant repeat analyses were performed with 27 individual samples of genomic DNA from human cells and tissues covering 30 – 60% of the domain. Within the peripheral regions, sequence variations 1– 4 occur at high incidence and show a nearly constant repeat topology in all individual normal and tumor samples. Also, individuals who were non-Caucasian or of different ethnic background were found to have the same set of replacements with identical topology. The repeat variant 1 replacing the established tumor target motif DTR with ESR was found in all individuals and appears predominantly in repeat clusters (diads and triads). The largely constant topology of variant repeats is interpreted by the assumption that the variable number of tandem repeats domain has evolved as a recent expansion of sequence variable super-repeats.

The human mucin MUC1 represents a well-established tumor marker in postoperative control of breast cancer patients and is currently being evaluated as a target in a variety of immunotherapeutic strategies including the development of efficient tumor vaccines (1–3). The immunodominant epitope of MUC1 is a short peptide motif (DTR) within the mucincharacteristic variable number of tandem repeats (VNTR)1 domain (4). This domain consists of variable numbers of a tandemly repeated icosapeptide and varies in length according to a genetic polymorphism (5, 6). The VNTR domain has been thought to be highly conserved with respect to the sequence of its peptide unit: PDTRPAPGSTAPPAHGVTSA (7–10). How* This study was supported by Deutsche Forschungsgemeinschaft Grant Ha2092/8-1 and Deutsche Krebshilfe Grant 70-2396-Ba I. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed. Tel.: 49-221-4784493; Fax: 49-221-478-6977; E-mail: [email protected]. 1 The abbreviations used are: MVRA, minisatellite variant repeat analysis; MVR, minisatellite variant repeat; VNTR, variable number of tandem repeats; PCR, polymerase chain reaction; bp, base pair(s). This paper is available on line at http://www.jbc.org

MATERIALS AND METHODS

Samples—Blood samples were obtained from five healthy unrelated individuals from the local blood bank. Breast tumor cell lines ZR75-1, 2

1

S. Mu¨ller and F.-G. Hanisch, unpublished observations.

AQ: R


2

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ARTNO: M103187200

Sequence Polymorphism of MUC1 Repeats

FIG. 1. a, 5⬘ and 3⬘ peripheral nucleotide sequences of the VNTR domain in MUC1 from T47D breast carcinoma cells. The entire VNTR domain was amplified by PCR, cloned, and sequenced. Sequence data obtained are underlined. This partial sequence information was combined with sequence data on single bands of the PCR product ladder, resulting in complete nucleotide sequences at both domain peripheries (the start points of the repeat units are indicated by arrows, and identified amino acid substitutions are indicated by bold letters). Sequence data on the flanking degenerate repeats were compared with the nucleotide sequences available under GenBankTM accession number M61170. b, sequence variations on the DNA and protein levels are identified in the human MUC1 VNTR domain.

AQ: F AQ: G

MCF-7, T47D, and MDA-MB231, colorectal cell lines HT-29 and LS174T, and gastric cell lines KATO-III and AGS were obtained from American Type Culture Collection (Manassas, VA). Breast cell line MTSV1-7 was a gift from Dr. J. Taylor-Papadimitriou (Imperial Cancer Research Fund, London, United Kingdom), whereas pancreatic cell lines PANC1 and S2-013 were a gift from Dr. M. A. Hollingsworth (Eppley Institute, University of Nebraska, Omaha, NE). Fresh tumor tissue samples were obtained from the Department of Surgery (University of Cologne, Cologne, Germany), snap-frozen in liquid nitrogen, and kept frozen at ⫺80 °C. Oligodeoxynucleotides—Two sets of oligodeoxynucleotides (flank5⬘VNTR and flank-3⬘VNTR) were designed from sequences flanking the 5⬘ end or the 3⬘ end of the MUC1-VNTR domain. Repeat-specific primers were synthesized complementary to the sequence variations (DT3 ES, P3 Q, P3 A, and P3 T) within the VNTR domain. The 5⬘ end of each MVR-specific primer carried a noncomplementary tail (N(20)). Oligodeoxynucleotides were synthesized by BioTeZ Berlin-Buch GmbH. The sequences of primers used for MVR-PCR are as follows: (a) N(20), 5⬘-TCCCgCgTCCATggCAgCTg (Conway et al., 1996); (b) flank5⬘VNTR, 5⬘-CTAgggggAAgAgAgTAgggAgAgggAAggC; (c) flank3⬘VNTR, 5⬘-gTgAgAgggAAAggACTCgggCTTgATg; (d) N(20)-DTR5⬘VNTR, 5⬘-N(20)-AgCCCggggCCggCCTggTg; (e) N(20)-DTR-3⬘VNTR,

5⬘-N(20)-TgTCACCTCggCCCCggACAC; (f) N(20)-ESR-5⬘VNTR, 5⬘N(20)-AgCCCggggCCggCCTgCTC; (g) N(20)-ESR-3⬘VNTR, 5⬘-N(20)-TgTCACCTCggCCCCggAgAg; (h) N(20)-P-3⬘VNTR, 5⬘-N(20)-gggCTCCACCgCCCCCCC; (i) N(20)-A-5⬘VNTR, 5⬘-N(20)-CgAggTgACACCgTgggCTgC; (j) N(20)-A-3⬘VNTR, 5⬘-N(20)-gggCTCCACCgCCCCCgC; (k) N(20)-Q-5⬘VNTR, 5⬘-N(20)-CgAggTgACACCgTgggCTTg; (l) N(20)-Q3⬘VNTR, 5⬘-N(20)-gggCTCCACCgCCCCCCA; (m) N(20)-T-5⬘VNTR, 5⬘N(20)-CgAggTgACACCgTgggCTgT; (n) N(20)-T-3⬘VNTR, 5⬘-N(20)-gggCTCCACCgCCCCCA; (o) SP6, 5⬘-gATTTAggTgACACTATAg; (p) T7, 5⬘TAATACgACTCACTATAggg; and (q) T3, 5⬘-ATTACCCCTCACTAAAgggA. DNA Isolation—Genomic DNA from the blood samples was prepared using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genomic DNA from cell lines and tissue samples was isolated using the Blood & Cell Culture DNA Kit (Qiagen). MVR Analyses—The MVR-PCR analysis performed in this study was based on the method described by Jeffreys et al. (18, 19). MVR analyses were performed by using a combination of MVR-specific primers and a primer fixed at the 5⬘ or 3⬘ sites in the DNA flanking the minisatellite to generate a ladder of PCR products starting from each variant repeat along the amplified alleles. Detection of minisatellite variant repeats and their amplification were uncoupled by providing a 5⬘ extension of 20


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3

TABLE I Topology of amino acid replacements at 5⬘ and 3⬘ peripheral regions in individual MUC1 VNTR domainsa Icosapeptide sequence: P D T R P A P G S T A P P A H G V T S A DNA

b

Repeats 5⬘-terminal of the VNTR-domain 1

01 02 03 04 05 06 07 08 09 10 AGS KATOIII T47D MCF-7 LS174T HT-29 ZR-75–1 MDA-MB231 MTSV1–7 PANC1 S2–013 11 pa-no 11 pa-ca 12 pa-ca 12 pa-no 13 co-ca 13 co-no 14 co-no 14 co-ca 15 ga-ca 15 ga-no 16 ga-ca 16 ga-no

2

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

3

4

5

6

7

3 2, 3 3 2, 3 3 3 1, 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

2 2 2 2 2 2 2, 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1, 2 1, 2

4 4

3 3 3 3 3 3 3

8

1 1

4 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 2, 3, 4 2, 3, 4 4 4

1 1

Repeats 3⬘-terminal of the VNTR-domain

9

10

1 1, 3 1 1, 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1, 3 1, 3 1 1 1 1 1 1

1, 3 1, 3 1, 3 3 1, 3 1, 3 3 1, 3 1, 3 1, 3 1, 3 3 1, 3 1, 3 1, 3 1, 3 1, 3 1, 3 1, 3 1, 4 1, 3 3 3 1, 3 1, 3 1 1, 3 1, 3 1, 3

1 1

1, 3 1, 3

11

1, 3

13

12

11

1 1

1 1

1 1 1 1 1 1

3 3

1

1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

10

9

8

7

6

5

4

3

2

1

3 1 1

1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1

1 1, 3, 4 1

1

1

1

1 1

1 1

1 1

1 1

a Four amino acid replacements within the repeat peptide of MUC1 VNTR domains were analyzed on the DNA level by MVRA of individual genomic samples. Numbers 1– 4 refer to the sequence variations: DT3 ES (GAC ACC3 GAG AGC), 1; P3 Q (CCA3 CAA), 2; P3 A (CCA3 GCA), 3; and P3 T (CCA3 ACA), 4. In repeats where nonvariant and variant sequences were detectable, the replacement number is given in italic. b Individual genomic DNA was obtained from blood cells. 01– 05, caucasians; 06, donor from Ghana; 07, donor from Tunisia; 08, donor from Iran; 09, donor from Morocco; and 10, donor from Indonesia. Tissue samples 11–16 were from cancer patients (no, normal; ca, carcinoma tissue) of pancreatic (pa), colorectal (co), or gastric (ga) origin.

AQ: H

bp (N(20)) to each MVR-specific oligodeoxynucleotide. Variant specific primers were used at a low concentration (50 nM), whereas the driving amplification passed with high concentration (1 ␮M) of the flanking primer of the VNTR domain and the N(20) primer itself. Samples (100 ng) of genomic DNA were amplified in 25-␮l reactions using the PCR buffer and enzyme system of GC-rich PCR Kit (Roche Diagnostics, Mannheim, Germany). Reactions were cycled once for 3 min at 95 °C, 40 s at 94 °C, and 1 min at 65 °C and for 4 min at 70 °C for 10 cycles on a DNA Thermal Cycler (Hybaid MBS; Hybaid, Heidelberg, Germany), followed by a chase for 40 s at 94 °C and 1 min at 65 °C, with 4 min and 5 s increment/cycle at 70 °C for 22 cycles, with a final extension of 10 min at 70 °C. The MVR-PCR product ladders were separated by electrophoresis in 1.4% MoSieve agarose gels (10 cm in length; peqLab, Erlangen, Germany) using TAE buffer (40 mM Tris acetate and 1 mM EDTA) and stained with ethidium bromide. The MVR-PCR product ladder starting at the 5⬘ or 3⬘ end of the VNTR domain was read from the bottom of the gels. The first repeat unit at the 5⬘ end of the domain was visible at approximately 650 bp, whereas the first repeat of the 3⬘ end was isographic with polynucleotides of approximately 550 bp. MVR analyses were performed for the 5⬘ and 3⬘ regions of the MUC1-VNTR with the appropriately designed primers. DNA Sequencing of PCR Products—Single bands within the PCR product ladders were cut from the agarose gel and purified using QiaEx Gel Extraction Kit (Qiagen). The cleared DNA samples were cloned into pGEM-T easy vector (Promega, Mannheim, Germany). After plasmid preparation using QIAprep Spin Miniprep Kit (Qiagen), DNA was sequenced using SP6 or T7 primer. The primer (10 pM) was annealed in the presence of 300 ng of plasmid DNA, 2 ␮l of the Big Dye Cycle Sequencing Ready Reaction Kit (PerkinElmer Life Sciences), and double distilled H2O in a 10-␮l reaction mixture under the following conditions: 2 min at 96 °C and 25 cycles of denaturation at 96 °C for 10 s and annealing at 55 °C for 4 min. The primer-annealed template was sequenced by the Big Dye terminator technique. After purification of

the sequenced samples, electrophoresis was performed on an automated sequencing machine (ABI 377; PerkinElmer Life Sciences). In this way, the sequencing of single bands within the PCR product ladder revealed the nucleotide sequence of 4 – 6 repeat units. By sequencing of overlapping regions of the domain periphery, it was possible to obtain the complete nucleotide sequence from the first 11 repeats of the 5⬘ end as well as of the first 13 repeat units of the 3⬘ end of the VNTR domain. These sequences could be aligned with those obtained by analysis of the 5⬘- and 3⬘-flanking regions accessible after amplification of the entire VNTR domain by primers annealing in the terminal regions of exon 2. Sequence information corroborated the data from MVRA with respect to the identity and topology of each of the four F1 sequence variations (Fig. 1, a and b). Length Polymorphism of Individual MUC1 VNTR Domains—The entire VNTR domain of MUC1 was amplified by PCR using a primer set flanking the VNTR domain (5⬘ primer, 5⬘-TgAgTATgACCAgCAgCgTACTCTCCAgCC; 3⬘ primer, 5⬘-ggAggTgAgAggAggTACCgTgCTATggTg). 200 ng of genomic DNA were amplified in 50-␮l reaction mixtures using the GC-rich PCR Kit (Roche Diagnostics). The temperature profile was as follows: once 3 min at 95 °C, 30 s at 95 °C, 4 min at 65 °C for AQ: I 10 cycles on a DNA Thermal Cycler (Biometra) followed by 30 s at 95 °C, 4 min ⫹ 5 s increment per each cycle at 65 °C for 25 cycles with a final extension of 10 min at 72 °C. The PCR products were separated by electrophoresis in 1% agarose gels and stained with ethidium bromide. In each sample, only one allele of the MUC1-VNTR domain was detectable. The number of repeat units that corresponded to the length of each individual allele was determined. To verify the identity of the amplified MUC1-VNTR domain, the band was cut from the gel, and the DNA was purified (QiaEx Gel Extraction Kit; Qiagen) and cloned into pCR XL-Topo vector (Topo XL PCR Cloning Kit; Invitrogen, Groningen, the Netherlands). The insert was cut out using restriction enzyme EcoRI (NEB, Frankfurt a.M., AQ: J Germany) and subcloned into pBluescript vector. After plasmid prepa-


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ration (Qiagen Spin Miniprep Kit; Qiagen), DNA was sequenced using T3 or T7 primers (ABI 377; PerkinElmer Life Sciences). Sequence data obtained were compared with the available nucleotide sequence of MUC1 (GenBankTM accession number M61170). At the 5⬘ end of the VNTR domain, legible sequence started in nucleotide position 3601. In position 3821, the tandem repeat unit started, and it was possible to read the sequence of two peripheral repeat units. Sequence data at the 3⬘ end of the domain started in nucleotide position 4181 and ended in position 3952 with the first degenerate repeat. RESULTS

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Four different sequence variations at the 5⬘- and 3⬘-terminal regions of the human MUC1 VNTR domain were analyzed by MVRA of 27 individual genomic DNA samples (Table I). The samples comprised normal tissues and cells (10 individual blood samples, 6 individual tissue samples adjacent to solid tumors, and 1 immortalized “normal” breast epithelial cell line) and a variety of cancer samples, including cell lines (2 gastric, 4 breast, 2 pancreatic, and 2 colorectal carcinoma cell lines) and solid tumors (2 pancreatic, 2 colorectal, and 2 gastric carcinomas). MVRA allowed for the identification of sequence variations in 11–13 terminal repeats from the 5⬘ and 3⬘ peripheries of the VNTR domain (Fig. 2, a and b). Amplification products containing larger numbers of repeats were visible in the gels, but unequivocal assignments could not be made. Hence, the reported relative frequencies of the occurrence of variant sequences do not represent the entire domain. The incidence of variant repeats was higher in the 5⬘-terminal region than in the 3⬘-terminal regions. Considering 11 repeats at the 5⬘-terminal region, about 54% of the repeats contain at least one of the four sequence variations, on average (Table I). Within 13 repeats at the 3⬘-terminal regions, the incidence is about 22%. It is obvious that some of the replacements, in particular, the DT3 ES substitution (sequence variation 1), occur in clusters (diads or triads), whereas others, like the P3 Q (sequence variation 2), P3 A (sequence variation 3), and P3 T (sequence variation 4), appear mainly in isolated repeats (Table I). To verify that the PCR band ladders from MVRA represented real sequence variations and not artifacts related to the primer design or PCR parameters, individual samples were analyzed by sequencing of the PCR products. By alignment of overlapping stretches (each corresponding to about 4 – 6 repeats) with partial sequences obtained after amplification of the entire VNTR domain, complete sequence information was revealed for 11–13 repeats of the peripheral regions. Data on human breast cancer cell line T47D are presented in Fig. 1a to place the new sequence information into the context of previous reports (7– 10) and to define where the numbering of repeats starts at the 5⬘- and 3⬘-flanking regions of the VNTR domain. An additional sequence variation, which was extracted from sequence information on a partial DNA corresponding to a pentarepeat of human MUC1 (6), the concerted replacement of His-Gly (CAC GGT) by Val-Arg (GTC CGT), was also analyzed by MVRA. Specifically designed detection primers indicated the occurrence of this sequence variation in a few individual samples at low frequency and with highly variable repeat topology. However, sequence analyses of amplified PCR products revealed that the replacement did not exist and that the primers had stochastically annealed. Sequence Variation 1: DT3 ES—The DT3 ES replacement (GAC ACC 3 GAG AGC) was found in all individual samples and represents the most frequent sequence variation within the peripheral regions of the VNTR domain (Table I). Within the 5⬘-terminal regions, sequence variation 1 is localized primarily in repeats 9 and 10 (Figs. 1 and 2). Nearly 90% of the individual samples contain this sequence variation in a repeat cluster (two or three adjacent repeats). A similar pattern is

FIG. 2. Minisatellite variant repeat mapping of the MUC1 VNTR domain. Genomic DNA from T47D breast cancer cells (a) and from individual blood sample 01 (b) was amplified by MVR-PCR. Products were separated by electrophoresis on 1.4% MoSieve agarose gel. Presentation was restricted to the variant repeat patterns. Gel band patterns are read from bottom to top, starting at the first positive band that corresponds to one of the alternative primer pairs (first repeat unit 5⬘-terminal, approximately 650 bp; first repeat unit 3⬘-terminal, approximately 550 bp). In each case, the MUC1 alleles could be read at least 11 (5⬘-terminal) or 13 (3⬘-terminal) repeat units into the minisatellite. The various replacements (DT3 ES, lanes 2 and 3; P3 T, lane 4; P3 Q, lane 5; P3 A, lane 6) within the icosapeptide of MUC1 show similar patterns in T47D breast cancer cells and in the blood cell sample.

revealed for the 3⬘-terminal region. All individual samples show a clustering from 2– 4 adjacent repeats, with the highest frequency in repeats 11 and 12. No evidence for a random distribution of this replacement in the terminal VNTR regions of individual MUC1 samples could be obtained. The topology


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FIG. 3. Representative topology of variant repeats within the peripheral regions of the MUC1 VNTR domain. Repeats with peptide sequences identical to the reference icosapeptide are indicated by white background areas. Variant repeats are shown with grayshaded areas, and the repeat-specific replacements are designated by the one-letter code for amino acids. Repeats in which only a subpopulation exhibited replacement (exceeding 25%) are indicated by striped areas.

and incidence of the DT3 ES replacement do not show strong individual fluctuations. Sequence Variations 2 and 3: P3 Q and P3 A—Both alternative variations occur at the same position within the repeat sequence by replacing Pro13 (CCA) with Q (CAA) or A (GCA), respectively. Their topology in the 5⬘ region is distinct but constant in all individual samples (Figs. 1 and 2; Table I). Accordingly, sequence variation 2 is restricted to repeats 2 and 6, whereas sequence variation 3 is confined mainly to repeats 5 and 10. With two exceptions, these variations were not detected in the 3⬘ regions of the VNTR domain. Sequence Variation 4: P3 T—The replacement of Pro13 (CCA) with Thr (ACA), which was detected in this study by sequencing of PCR products, introduces a new potential glycosylation site into the repeat. It has not been described previously and seems to occur at low incidence. We could detect it exclusively in repeat 7 (5⬘ region) (Figs. 1 and 2; Table I) with the exception of AGS cells, in which the first repeat of the 3⬘ region was positive for this variant. Interestingly, analysis of the individual lengths of VNTR domains revealed that the occurrence of this variant was negatively associated with higher numbers of repeats (see below). A representative pattern of sequence variations is shown for the 5⬘ and 3⬘ regions in Fig. 3. This pattern represents the majority of the individual samples and hence indicates the nonrandom distribution of sequence variations 1– 4. No differences with respect to the topology or incidence of the sequence variations in tumor versus normal samples became evident. Also individuals of non-Caucasian race or different ethnic background were found to have the same set of replacements and, except for one individual, identical topology. Analysis of Length Polymorphism—In 20 individual samples, MUC1 allele lengths ranged from 40 –52 tandemly repeated 60-bp units, whereas 60 – 84 repeat units were found in 5 individual samples. In 18 of the 20 individuals, who have a smaller MUC1 allele, the substitution of P3 T (variant 4) in repeat 7 (5⬘ peripheral region of the VNTR domain) was detectable. Only two individuals in the group did not show this replacement. By contrast, in all individual domains that exhibit more than 60 repeat units, a P3 T substitution was not identified in the respective repeat. The length determination of individual MUC1 alleles allowed us to estimate that 30 – 60% of the VNTR domain was covered by MVRA. DISCUSSION

The present study reveals for the first time insight into the nucleotide sequences of peripheral regions in the human MUC1 repeat domain. Although the primary structure of this mucin was elucidated about a decade ago, the entire domain of repetitive 60 bp comprising between 20 and 120 units was never subjected to a detailed sequence analysis. Repetitive DNA se-

quences, in particular, GC-rich polynucleotides, are difficult to amplify by standard PCR protocols. Accordingly, only partial information was previously accessible after amplification of the entire domain in exon 2 and sequence analysis of a few terminal repeats. The published repeat sequences from four independent groups (7–10) agree in the finding that the sequence of a common 60-bp unit does not show structural variation in different cellular specimens. In contrast to this, we recently obtained evidence on the protein level that individual domains can display considerable sequence variation reflected in a series of amino acid replacements in the 20-amino acid repeat (11). The singular observation was demonstrated in the present study to represent a general phenomenon by showing that all individual MUC1 alleles under study do contain the same pattern of sequence variations with largely identical topology. The nearly constant localization of variant repeats in the peripheral regions indicates that they may have originated before multiple duplications generated a length polymorphic VNTR domain. The entire domain may have evolved accordingly as a recent expansion of sequence variable super-repeats. Sequence variability of repeat peptides seems to be a common feature of mucins because other members of the MUC family (MUC4) also exhibit multiple replacements in their repetitive units (20). Sequence variability raises the question of whether functional aspects of the mucin domain may be affected. However, because only one repeat in MUC1 was demonstrated to contain a further potential glycosylation site, whereas all other replacements preserve the site pattern, it can be assumed that the sequence context-dependent initiation of O-glycosylation should not be influenced. Semiquantitative Edman analyses of repeat peptides from tumor-associated MUC1 have confirmed this assumption and, in particular, did not reveal any indication of altered O-glycosylation of the variant ESR motif (11). On the other hand, in vitro studies have recently shown that the replacement of Pro13 with Ala in the repeat peptide AHG21 has a strong negative effect on initial O-glycosylation catalyzed by AQ: N rGalNAc-T2 (21). Variations of the peptide sequence are expected to have a strong influence on the conformation and hence the antigenicity of the repeat unit. The most frequent variation, DT3 ES (sequence variation 1), could be of importance in the context of tumor vaccination because it may represent an alternative target for humoral or cellular immune responses. Some murine monoclonal antibodies with a specificity for the DTR motif are cross-reactive to this variant motif, whereas the majority are not.3 This indicates that the ESR motif exhibits distinct struc- Fn3 tural features, despite the fact that the amino acid replacements are conservative. To reveal the conformational features 3

F.-G. Hanisch, unpublished observations.


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REFERENCES

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of the variant motif and its glycosylated derivatives, synthetic (glyco)peptides are currently analyzed by 800 MHz NMR spectroscopy. Knowledge of the structural features of the two alternative motifs (DTR versus ESR) in the repeat peptide of MUC1 is expected to aid the development of efficient tumor vaccines.

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1. Bon, G. G., Kenemans, P., van Kamp, G. J., Yedema, C. A., and Hilgers, J. (1990) J. Nucl. Allied Sci. 34, 151–162 2. von Mensdorff-Pouilly, S., Gourevitch, M. M., Kenemans, P., Verstraeten, A. A., Litvinov, S. V., van Kamp, G. J., Meijer, S., Vermorken, J., and Hilgers, J. (1996) Eur. J. Cancer 32A, 1325–1331 3. von Mensdorff-Pouilly, S., Petrakou, E., Kenemans, P., van Uffelen, K., Verstraeten, A. A., Snijdewint, F. G., van Kamp, G. J., Schol, D. J., Reis, C. A., Price, M. R., Livingston, M. O., and Hilgers, J. (2000) Int. J. Cancer 86, 702–712 4. Price, M. R., Rye, P. D., Petrakou, E., Murray, A., Brady, K., Imai, S., Haga, S., Kiyozuka, Y., Schol, D., Meulenbroek, M. F., et al. (1998) Tumour Biol. 19, 1–20 5. Gendler, S. J., Taylor-Papadimitriou, J., Duhig, T., Rothbard, J., and Burchell, J. (1988) J. Biol. Chem. 263, 12820 –12823 6. Siddiqui, J., Abe, M., Hayes, D., Shani, E., Yunis, E., and Kufe, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2320 –2323 7. Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J., Pemberton, L., Lalani, E., and Wilson. (1990) J. Biol. Chem. 265, 15286 –15293 8. Lan, M. S., Batra, S. K., Qui, W.-N., Metzgar, R. S., and Hollingsworth, M. A.

(1990) J. Biol. Chem. 265, 15294 –15299 9. Ligtenberg, M. J. L., Vos, H. L., Gennissen, A. M. C., and Hilkens, J. (1990) J. Biol. Chem. 265, 5573–5578 10. Wreschner, D. H., Hareuveni, M., Tsarfaty, I., Smorodinsky, N., Horev, J., Zaretzky, J., Kotkes, P., Weiss, M., Lathe, R., Dion, A., and Keydar, I. (1990) Eur. J. Biochem. 189, 463– 473 11. Mu¨ller, S., Alving, K., Peter-Katalinic, J., Zachara, N., Gooley, A. A., and Hanisch, F.-G. (1999) J. Biol. Chem. 274, 18165–18172 12. Hanisch, F.-G., and Mu¨ller, S. (2000) Glycobiology 10, 439 – 449 13. Jerome, K. R., Barud, D. L., Bendt, K. M., Boyer, C. M., Taylor-Papdimitriou, J., McKenzie, I. F. C., Bast, R. C., and Finn, O. J. (1991) Cancer Res. 51, 2908 –2916 14. Apostolopoulos, V., Loveland, B. E., Pietersz, G. A., and McKenzie, I. F. C. (1995) J. Immunol. 155, 5089 –5094 15. Apostolopoulos, V., Karanikas, V., Haurum, J. S., and McKenzie, I. F. C. (1997) J. Immunol. 159, 5211–5218 16. Agrawal, B., Reddish, M. A., and Longenecker, B. M. (1996) J. Immunol. 157, 2089 –2095 17. Taylor-Papadimitriou, J., Burchell, J., Miles, D. W., and Dalziel, M. (1999) Biochim. Biophys. Acta 1455, 301–313 18. Jeffreys, A. J., Neumann, R., and Wilson, V. (1990) Cell 60, 473– 485 19. Jeffreys, A. J., MacLeod, A., Tamaki, K., Neil, D. L., and Monckton, D. G. (1991) Nature 354, 204 –209 20. Porchet, N., van Cong, N., Dufosse, J., Audie, J. P., Guyonnet-Duperat, V., Gross, M. S., Denis, C., Degand, P., Bernheim, A., and Aubert, J. P. (1991) Biochem. Biophys. Res. Commun. 175, 414 – 422 21. Hanisch, F.-G., Reis, C., Clausen, H., and Paulsen, H. (2001) Glycobiology, in press

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AUTHOR QUERIES AUTHOR PLEASE ANSWER ALL QUERIES

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A—Au: Changes to the first two sentences of the summary OK? B—Au: Change as meant (‘individuals who were non-Caucasian or of different ethnic background‘)? C—Au: The Journal guidelines state that the summary should be no longer than 200 words. At this stage, we only require that the summary fit into the left-hand column. If your summary is long enough to run over into the right hand column, please cut text. D—Au: Change as meant (‘No nucleotide sequence information was reported. . . ‘)? If not, please rewrite this sentence for clarity. E—Au: If HMFG1 is an abbreviation and not a designation, please write out. F—Au: Please explain the significance of the use of uppercase and lowercase letters in the sequences of primers. G—Au: No work by Conway et al. (see ‘Oligonucleotides‘ in ‘Materials and Methods‘) is listed in the references. If you are referring to a published article, please add Conway et al to the reference list as ref. 22 and cite ref. 22 here (DO NOT RENUMBER THE REFERENCES). If you are referring to unpublished information, cite footnote 3 here and provide the necessary information for footnote 3. H—Au: Changes OK (see the sentence beginning ‘Reactions were cycled once for 3 min at 95..‘)? If not, please rewrite this sentence for clarity. I—Au: Please rewrite the remainder of this sentence for clarity (‘The temperature profile was as follows:. . . ‘). J—Au: If NEB and a.M. are abbreviations, please write out. K—Au: genebank changed to GenBank throughout. Change OK? L—Au: Please check the italic numbers in Table I carefully to ensure they are correct as set. Please explain the significance of the bold residues in the table. M—Au: Change as meant (‘and not artifacts related to primer design‘)? N—Au: If rGalNAc-T2 is an abbreviation, please write out.

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AUTHOR QUERIES AUTHOR PLEASE ANSWER ALL QUERIES O—Au: Journal in ref. 1 is not listed in our sources. Please confirm that the journal title is correct as set. P—Au: Please provide initials for the last author in ref. 7. Q—Au: Please provide volume number and page range for ref. 21, if known. R—Au: Please confirm that author initials have been added as meant for footnote 2. S—Au: Changes to last sentence of Fig. 3 legend OK (‘Repeats in which only a subpopulation exhibited replacement. . . ‘)?

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