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• W1966641794 abstract "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 DT→ES (sequence variation 1) and the single replacements P→Q (sequence variation 2), P→A (sequence variation 3), and P→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 5′ and 3′ 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. 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 DT→ES (sequence variation 1) and the single replacements P→Q (sequence variation 2), P→A (sequence variation 3), and P→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 5′ and 3′ 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. variable number of tandem repeats minisatellite variant repeat analysis minisatellite variant repeat polymerase chain reaction base pair(s) 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 (1Tondini C. Hayes D.F. Gelman R. Henderson I.C. Kūfe D.W. Cancer Res. 1988; 48: 4107-4112PubMed Google Scholar, 2von Mensdorff-Pouilly S. Gourevitch M.M. Kenemans P. Verstraeten A.A. Litvinov S.V. van Kamp G.J. Meijer S. Vermorken J. Hilgers J. Eur. J. Cancer. 1996; 32A: 1325-1331Abstract Full Text PDF PubMed Scopus (105) Google Scholar, 3von 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. Hilgers J. Int. J. Cancer. 2000; 86: 702-712Crossref PubMed Google Scholar). The immunodominant epitope of MUC1 is a short peptide motif (DTR) within the mucin-characteristic variable number of tandem repeats (VNTR)1 domain (4Price M.R. Rye P.D. Petrakou E. Murray A. Brady K. Imai S. Haga S. Kiyozuka Y. Schol D. Meulenbroek M.F. et al.Tumour Biol. 1998; 19: 1-20Crossref PubMed Scopus (123) Google Scholar). This domain consists of variable numbers of a tandemly repeated icosapeptide and varies in length according to a genetic polymorphism (5Gendler S.J. Taylor-Papadimitriou J. Duhig T. Rothbard J. Burchell J. J. Biol. Chem. 1988; 263: 12820-12823Abstract Full Text PDF PubMed Google Scholar, 6Siddiqui J. Abe M. Hayes D. Shani E. Yunis E. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2320-2323Crossref PubMed Scopus (278) Google Scholar). The VNTR domain has been thought to be highly conserved with respect to the sequence of its peptide unit: PDTRPAPGSTAPPAHGVTSA (7Gendler S.J. Lancaster C.A. Taylor-Papadimitriou J. Duhig T. Peat N. Burchell J. Pemberton L. Lalani E. Wilson D. J. Biol. Chem. 1990; 265: 15286-15293Abstract Full Text PDF PubMed Google Scholar, 8Lan M.S. Batra S.K. Qui W.-N. Metzgar R.S. Hollingsworth M.A. J. Biol. Chem. 1990; 265: 15294-15299Abstract Full Text PDF PubMed Google Scholar, 9Ligtenberg M.J.L. Vos H.L. Gennissen A.M.C. Hilkens J. J. Biol. Chem. 1990; 265: 5573-5578Abstract Full Text PDF PubMed Google Scholar, 10Wreschner D.H. Hareuveni M. Tsarfaty I. Smorodinsky N. Horev J. Zaretzky J. Kotkes P. Weiss M. Lathe R. Dion A. Keydar I. Eur. J. Biochem. 1990; 189: 463-473Crossref PubMed Scopus (206) Google Scholar). However, 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 (11Müller S. Alving K. Peter-Katalinic J. Zachara N. Gooley A.A. Hanisch F.-G. J. Biol. Chem. 1999; 274: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The concerted replacement of Asp2-Thr3 → 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 → Ala was demonstrated to occur in approximately 30% of the repeats (11Müller S. Alving K. Peter-Katalinic J. Zachara N. Gooley A.A. Hanisch F.-G. J. Biol. Chem. 1999; 274: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 12Hanisch F.-G. Müller S. Glycobiology. 2000; 10: 439-449Crossref PubMed Scopus (231) Google Scholar). Evidence for variations on the protein level was also revealed for MUC1 from individual human milk samples. 2S. Müller and F.-G. Hanisch, unpublished observations.2S. Müller and F.-G. Hanisch, unpublished observations. No nucleotide sequence information was reported to confirm these 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 (4Price M.R. Rye P.D. Petrakou E. Murray A. Brady K. Imai S. Haga S. Kiyozuka Y. Schol D. Meulenbroek M.F. et al.Tumour Biol. 1998; 19: 1-20Crossref PubMed Scopus (123) Google Scholar) and also in humans (2von Mensdorff-Pouilly S. Gourevitch M.M. Kenemans P. Verstraeten A.A. Litvinov S.V. van Kamp G.J. Meijer S. Vermorken J. Hilgers J. Eur. J. Cancer. 1996; 32A: 1325-1331Abstract Full Text PDF PubMed Scopus (105) Google Scholar) and represents the primary target site for major histocompatibility complex-unrestricted cytotoxic T-cell responses in breast cancer patients (13Jerome K.R. Barud D.L. Bendt K.M. Boyer C.M. Taylor-Papdimitriou J. McKenzie I.F.C. Bast R.C. Finn O.J. Cancer Res. 1991; 51: 2908-2916PubMed Google Scholar). Classical major histocompatibility complex-restricted responses to epitopes within the tandem repeat have been reported for many mouse strains (14Apostolopoulos V. Loveland B.E. Pietersz G.A. McKenzie I.F.C. J. Immunol. 1995; 155: 5089-5094PubMed Google Scholar), and human cytotoxic T lymphocytes recognizing MUC1 peptide epitopes were also demonstrated (15Apostolopoulos V. Karanikas V. Haurum J.S. McKenzie I.F.C. J. Immunol. 1997; 159: 5211-5218PubMed Google Scholar), including major histocompatibility complex class II-dependent helper T-cell responses in vitro(16Agrawal B. Reddish M.A. Longenecker B.M. J. Immunol. 1996; 157: 2089-2095PubMed Google Scholar). 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 peritoneum of ovarian cancer patients, and these initial trials were recently extended in a Phase III multicenter clinical trial (17Taylor-Papadimitriou J. Burchell J. Miles D.W. Dalziel M. Biochim. Biophys. Acta. 1999; 1455: 301-313Crossref PubMed Scopus (419) Google Scholar). 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 (17Taylor-Papadimitriou J. Burchell J. Miles D.W. Dalziel M. Biochim. Biophys. Acta. 1999; 1455: 301-313Crossref PubMed Scopus (419) Google Scholar). 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 (17Taylor-Papadimitriou J. Burchell J. Miles D.W. Dalziel M. Biochim. Biophys. Acta. 1999; 1455: 301-313Crossref PubMed Scopus (419) Google Scholar). 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 (7Gendler S.J. Lancaster C.A. Taylor-Papadimitriou J. Duhig T. Peat N. Burchell J. Pemberton L. Lalani E. Wilson D. J. Biol. Chem. 1990; 265: 15286-15293Abstract Full Text PDF PubMed Google Scholar, 8Lan M.S. Batra S.K. Qui W.-N. Metzgar R.S. Hollingsworth M.A. J. Biol. Chem. 1990; 265: 15294-15299Abstract Full Text PDF PubMed Google Scholar, 9Ligtenberg M.J.L. Vos H.L. Gennissen A.M.C. Hilkens J. J. Biol. Chem. 1990; 265: 5573-5578Abstract Full Text PDF PubMed Google Scholar, 10Wreschner D.H. Hareuveni M. Tsarfaty I. Smorodinsky N. Horev J. Zaretzky J. Kotkes P. Weiss M. Lathe R. Dion A. Keydar I. Eur. J. Biochem. 1990; 189: 463-473Crossref PubMed Scopus (206) Google Scholar), are of utmost importance for the design of efficient tumor vaccines. Blood samples were obtained from ten healthy unrelated individuals from the local blood bank. Breast tumor cell lines ZR75-1, 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. Two sets of oligodeoxynucleotides (flank-5′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 (DT→ES, P→Q, P→A, and P→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 (22Conway K. Edmiston S.N. Hūlka B.S. Garrett P.A. Liū E.T. Cancer Res. 1996; 56: 4773-4777PubMed Google Scholar); (b) flank-5′VNTR, 5′-CTAgggggAAgAgAgTAgggAgAgggAAggC; (c) flank-3′VNTR, 5′-gTgAgAgggAAAggACTCgggCTTgATg; (d) N(20)-DTR-5′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-5′VNTR, 5′-N(20)CgAggTgACACCgTgggCTgg; (i) N(20)-P-3′VNTR, 5′-N(20)-gggCTCCACCgCCCCCCC; (j) N(20)-A-5′VNTR, 5′-N(20)-CgAggTgACACCgTgggCTgC; (k) N(20)-A-3′VNTR, 5′-N(20)-gggCTCCACCgCCCCCgC; (l) N(20)-Q-5′VNTR, 5′-N(20)-CgAggTgACACCgTgggCTTg; (m) N(20)-Q-3′VNTR, 5′-N(20)-gggCTCCACCgCCCCCCA; (n) N(20)-T-5′VNTR, 5′-N(20)-CgAggTgACACCgTgggCTgT; (o) N(20)-T-3′VNTR, 5′-N(20)-gggCTCCACCgCCCCCA; (p) SP6, 5′-gATTTAggTgACACTATAg; (q) T7, 5′-TAATACgACTCACTATAggg; and (r) T3, 5′-ATTACCCCTCACTAAAgggA. 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). The MVR-PCR analysis performed in this study was based on the method described by Jeffreys et al. (18Jeffreys A.J. Neumann R. Wilson V. Cell. 1990; 60: 473-485Abstract Full Text PDF PubMed Scopus (354) Google Scholar,19Jeffreys A.J. MacLeod A. Tamaki K. Neil D.L. Monckton D.G. Nature. 1991; 354: 204-209Crossref PubMed Scopus (326) Google Scholar). 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 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 µm) of the flanking primer of the VNTR domain and the N(20) primer itself. Samples (100 ng) of genomic DNA were amplified in 25-µl reactions using the PCR buffer and enzyme system of GC-rich PCR Kit (Roche Diagnostics, Mannheim, Germany). Reaction mixtures were incubated for 3 min at 95 °C, and then cycled for 40 s at 94 °C, 1 min at 65 °C and 4 min at 70 °C (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. 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 µl of the Big Dye Cycle Sequencing Ready Reaction Kit (PerkinElmer Life Sciences), and double distilled H2O in a 10-µl 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 sequence variations (Fig. 1,a and b). 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-µl reaction mixtures using the GC-rich PCR Kit (Roche Diagnostics). The temperature profile was as follows: once 3 min at 95 °C, cycling for 30 s at 95 °C, 4 min at 65 °C (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 (New England BioLabs, Frankfurt a.M., Germany) and subcloned into pBluescript vector. After plasmid preparation (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 numberM61170). 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. 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 (TableI). 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 andb). 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 DT→ES substitution (sequence variation 1), occur in clusters (diads or triads), whereas others, like the P→Q (sequence variation 2), P→A (sequence variation 3), and P→T (sequence variation 4), appear mainly in isolated repeats (TableI).Table ITopology of amino acid replacements at 5′ and 3′ peripheral regions in individual MUC1 VNTR domains1-aFour 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: DT→ES (GAC ACC→GAG AGC), 1; P→Q (CCA→CAA), 2; P→A (CCA→GCA), 3; and P→T (CCA→ACA), 4. In repeats where nonvariant and variant sequences were detectable, the replacement number is given in italic.DNA1-bIndividual 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.Icosapeptide sequence: P D T R P A P G S T A P P A H G V T S ARepeats 5′-terminal of the VNTR-domainRepeats 3′-terminal of the VNTR-domain12345678910111312111098765432101232411, 3110222, 32411, 31, 3110323211, 31, 31130422, 3211, 33110523211, 311106232411, 3110721, 32, 33330823211, 3311109232411, 31110232411, 311AGS232411, 3111, 3, 4KATOIII23241311T47D232411, 31111MCF-7232411, 311LS174T232411, 31111HT-29232411, 311ZR-75–1232411, 3111MDA-MB23123211, 3111MTSV1–723211, 3111PANC1232411, 411S2–013232411, 31111 pa-no232411,331111 pa-ca232411,331112 pa-ca23211, 31112 pa-no23211, 31113 co-ca224111113 co-no232411, 31114 co-no23211, 311114 co-ca23211, 311115 ga-ca231, 22, 3, 41115 ga-no231, 22, 3, 41116 ga-ca23411, 3111116 ga-no23411, 311111-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: DT→ES (GAC ACC→GAG AGC), 1; P→Q (CCA→CAA), 2; P→A (CCA→GCA), 3; and P→T (CCA→ACA), 4. In repeats where nonvariant and variant sequences were detectable, the replacement number is given in italic.1-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. Open table in a new tab 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. 1 a to place the new sequence information into the context of previous reports (7Gendler S.J. Lancaster C.A. Taylor-Papadimitriou J. Duhig T. Peat N. Burchell J. Pemberton L. Lalani E. Wilson D. J. Biol. Chem. 1990; 265: 15286-15293Abstract Full Text PDF PubMed Google Scholar, 8Lan M.S. Batra S.K. Qui W.-N. Metzgar R.S. Hollingsworth M.A. J. Biol. Chem. 1990; 265: 15294-15299Abstract Full Text PDF PubMed Google Scholar, 9Ligtenberg M.J.L. Vos H.L. Gennissen A.M.C. Hilkens J. J. Biol. Chem. 1990; 265: 5573-5578Abstract Full Text PDF PubMed Google Scholar, 10Wreschner D.H. Hareuveni M. Tsarfaty I. Smorodinsky N. Horev J. Zaretzky J. Kotkes P. Weiss M. Lathe R. Dion A. Keydar I. Eur. J. Biochem. 1990; 189: 463-473Crossref PubMed Scopus (206) Google Scholar) 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 (6Siddiqui J. Abe M. Hayes D. Shani E. Yunis E. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2320-2323Crossref PubMed Scopus (278) Google Scholar), 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. The DT→ES replacement (GAC ACC → 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 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 and incidence of the DT→ES replacement do not show strong individual fluctuations. 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. 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. 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 P→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 P→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. 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 sequences, 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 (7Gendler S.J. Lancaster C.A. Taylor-Papadimitriou J. Duhig T. Peat N. Burchell J. Pemberton L. Lalani E. Wilson D. J. Biol. Chem. 1990; 265: 15286-15293Abstract Full Text PDF PubMed Google Scholar, 8Lan M.S. Batra S.K. Qui W.-N. Metzgar R.S. Hollingsworth M.A. J. Biol. Chem. 1990; 265: 15294-15299Abstract Full Text PDF PubMed Google Scholar, 9Ligtenberg M.J.L. Vos H.L. Gennissen A.M.C. Hilkens J. J. Biol. Chem. 1990; 265: 5573-5578Abstract Full Text PDF PubMed Google Scholar, 10Wreschner D.H. Hareuveni M. Tsarfaty I. Smorodinsky N. Horev J. Zaretzky J. Kotkes P. Weiss M. Lathe R. Dion A. Keydar I. Eur. J. Biochem. 1990; 189: 463-473Crossref PubMed Scopus (206) Google Scholar) 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 (11Müller S. Alving K. Peter-Katalinic J. Zachara N. Gooley A.A. Hanisch F.-G. J. Biol. Chem. 1999; 274: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). 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 (20Porchet N. van Cong N. Dufosse J. Audie J.P. Guyonnet-Duperat V. Gross M.S. Denis C. Degand P. Bernheim A. Aubert J.P. Biochem. Biophys. Res. Commun. 1991; 175: 414-422Crossref PubMed Scopus (327) Google Scholar). 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 (11Müller S. Alving K. Peter-Katalinic J. Zachara N. Gooley A.A. Hanisch F.-G. J. Biol. Chem. 1999; 274: 18165-18172Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). 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 recombinant UDP-galNAc polypeptide N-acetylgalactosaminyl-transferase galNAc-T2 (21Hanisch, F.-G., Reis, C., Clausen, H., and Paulsen, H. (2001)Glycobiology, in press.Google Scholar). 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, DT→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. 3F.-G. Hanisch, unpublished observations. This indicates that the ESR motif exhibits distinct structural features, despite the fact that the amino acid replacements are conservative. To reveal the conformational features 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." @default.
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• W1966641794 title "Identification and Topology of Variant Sequences within Individual Repeat Domains of the Human Epithelial Tumor Mucin MUC1" @default.
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