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Originally published In Press as doi:10.1074/jbc.M406943200 on December 28, 2004

J. Biol. Chem., Vol. 280, Issue 11, 10655-10663, March 18, 2005
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A Novel Protein Derived from the MUC1 Gene by Alternative Splicing and Frameshifting*

Fiana Levitin{ddagger}§, Amos Baruch{ddagger}§, Mordechai Weiss¶, Keren Stiegman{ddagger}, Mor-li Hartmann{ddagger}, Merav Yoeli-Lerner{ddagger}, Ravit Ziv{ddagger}, Sheila Zrihan-Licht{ddagger}, Sima Shina{ddagger}, Andrea Gat||, Beatrice Lifschitz||, Moshe Simha**, Yona Stadler**, Alina Cholostoy{ddagger}, Benny Gil{ddagger}, David Greaves{ddagger}{ddagger}, Iafa Keydar{ddagger}, Joseph Zaretsky{ddagger}, Nechama Smorodinsky{ddagger}, and Daniel H. Wreschner{ddagger}§§

From the {ddagger}Department of Cell Research and Immunology, Tel-Aviv University, Ramat Aviv 69978, Israel, ||Department of Pathology, Sourasky-Ichilov Medical Center, Tel-Aviv 64239, Israel, **Surgical Department A, Sourasky-Ichilov Medical Center, Tel-Aviv 64239, Israel, Department of Endocrinology, Assaf Harofe Medical Center, Tzrifin 70300, Israel, and {ddagger}{ddagger}Department of Pathology, South Parks Road, Oxford University, Oxford OX1RE, United Kingdom

Received for publication, June 22, 2004 , and in revised form, December 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Genes that have been designated the name "MUC" code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Genes classified, for the most part, as MUC genes code for proteins that comprise mucin domains rich in proline, threonine, and serine residues (1). The heavily glycosylated mucin proteins derived from the MUC genes can be divided into those that are secreted from the cell (secreted mucins) and mucins that comprise a transmembrane domain that anchors them to the cell membrane (transmembrane mucins). The archetype membrane-tethered mucin, which was the one to be first characterized and sequenced, is derived from the MUC1 gene (2–5). It is a transmembrane protein that contains the serine-threonine-rich tandem-repeat region in its extracellular domain (Fig. 1A, MUC1/REP) and also comprises a 72 amino acid tail that can be tyrosine-phosphorylated (6, 7). The phosphorylated cytoplasmic domain subsequently interacts with second messenger proteins (6–23), thereby relaying a signal to the nucleus that modifies gene expression.



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  		FIG. 1.
Scheme of different MUC1 isoforms. A, the splicing events that generate various MUC1 mRNA isoforms are presented. MUC1/ZD mRNA is generated by alternative splicing that utilizes the same splice donor site as that utilized by the MUC1/Y isoform. The MUC1/ZD splice acceptor site (*S.A.) is located 19 nucleotides downstream to that used by MUC1/Y (see B for splice site locations). The alternate splice acceptor site *S.A. effects a reading frameshift depicted by stippled exons downstream to this site. B, MUC1/ZD nucleotide and protein sequence. Numbering of nucleotide and amino acids appear to the left and right of the figure, respectively. The splice event that generates MUC1/ZD mRNA and protein is indicated by the downward facing arrow just C-terminal to amino acid number 53 (after amino acid sequence EKNA). Signal peptide cleavage is predicted to occur between Gly23 and Ser24 (indicated by upward facing red arrow). MUC1/ZD thus will have 30 N-terminal amino acid sequences (stretching from Ser24-Ala53) in common with other MUC1 isoforms. The subsequent 43 C-terminal amino acids are unique to MUC1/ZD.

 
Challenging the classical definition of a mucin gene is the discovery of MUC1 mRNAs, which although transcribed from a mucin gene are devoid of a tandem-repeat array (24–32). Alternative splicing generates these MUC1-derived mRNAs that no longer code for tandem-repeat array-containing mucin proteins. The prime example is the tandem-repeat array-deleted MUC1/Y isoform, which is expressed both as mRNA and protein (24–26, 30, 31). The MUC1/Y protein is similar to MUC1/REP in that it also contains the transmembrane and cytoplasmic domains, yet it lacks the central tandem-repeats and their flanking sequences (Fig. 1A). Further studies reveal another MUC1 isoform, MUC1/X, that is also devoid of the tandemrepeat array (29). This isoform is generated by an alternative splicing event that has the same splice donor site as MUC1/Y but utilizes an alternative splice acceptor site located 54 nucleotides upstream to that used by MUC1/Y. Downstream to the splice acceptor sites, the MUC1/Y and MUC1/X isoforms retain the same reading frame as the repeat array-containing MUC1 transmembrane protein (29). Additional MUC1 mRNA splice forms have the potential to generate MUC1 gene-derived proteins that differ at their N and C termini (27, 28). The splice forms that alter the predicted site of signal peptide cleavage generate N-terminal diversity, whereas splice forms that either truncate or alter the reading frame of the cytoplasmic domain accomplish C-terminal diversity. Alternative splicing may also generate MUC1 proteins that are secreted from the cell rather than being tethered at the cell membrane (4, 31). Numerous splice variants have also been proposed for MUC4 (33).

We report here an alternatively spliced MUC1 mRNA that deletes the central tandem-repeat array. Following the discovery of the tandem-repeat array lacking the MUC1/Y isoform, this finding is not totally unexpected. What is surprising is the fact that downstream to the splice acceptor site, the usual MUC1 reading frame has undergone a +1 reading frameshift. This creates the potential for a novel MUC1 protein, designated here as MUC1/ZD, that comprises a completely new C-terminal protein sequence different from all of the other MUC1 proteins (MUC1/ZD GenBankTM accession numbers AY466157 [GenBank] (locus) and AAR28764 [GenBank] 1 (protein)). The existence of this MUC1 isoform as an expressed protein has not been previously documented. Here we show the expression at the protein level of a frameshifted MUC1 protein, the MUC1/ZD isoform.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
RT-PCR of MUC1 mRNA—Poly(A)-rich RNA extracted from breast cancer cells was reverse-transcribed (SuperscriptII, Invitrogen) using the reverse antisense oligonucleotide primer 5'-TGGCACATCACTCACGCTGACGTCTGAGATC-3' (complementary to nucleotides 1243–1272 in the MUC1/REP sequence, for numbering see Fig. 3B in Ref. 4) and 10% reverse transcription (RT)1 reaction subjected to PCR amplification (AmpliTaq Gold, Applied BioSystems) with the same reverse antisense primer and the forward sense oligonucleotide primer 5'-GAATCTGTTCTGCCCCCTCCCCAC-3' (nucleotides 18–42). 35 PCR cycles of 92 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s were employed. MUC1-specific RT-PCR products were subcloned into M13, and sequencing was accomplished using the dideoxynucleotide chain termination method.

Cloning of MUC1/ZD—For plasmid propagation in bacteria, the MUC1/ZD cDNA (Fig. 1) was subcloned into the ppolyII vector (34).

Cloning of MUC1/ZD in Glutathione S-Transferase (GST) Expression Vector—DNA coding for MUC1/ZD was inserted into the pGEX-2T bacterial expression vector (Amersham Biosciences). The resulting pGEX-2T-MUC1/ZD plasmid codes for a protein that comprises (from its N terminus) GST followed by a thrombin cleavage site, the MUC1/ZD sequence, and terminates with a protein kinase A phosphorylation site.

Purification of Recombinant Bacterial GST-MUC1/ZD Fusion Protein—Isopropyl 1-thio-{beta}-D-galactopyranoside-induced bacteria were lysed by sonication, and the recombinant bacterial GST-MUC1/ZD fusion protein was purified from the supernatant using glutathione-agarose beads (Amersham Biosciences) as described by the manufacturer. When pure MUC1/ZD protein was required, the GST fusion protein was incubated with thrombin (1 cleavage unit/100 µg) for 16 h at 25 °C.

Antibodies: Rabbit Polyclonal Anti-MUC1/ZD Antibodies—Three female rabbits were immunized with 200 µg of recombinant GST-MUC1/ZD protein mixed with adjuvant. Affinity-purified anti-MUC1/ZD antibodies were obtained using a MUC1/ZD affinity column prepared with Affi-Gel-10 beads (Bio-Rad).

Mouse Monoclonal Anti-MUC1/ZD Antibodies—The purified recombinant MUC1/ZD protein was first coupled to keyhole limpet hemocyanin. The MUC1/ZD-keyhole limpet hemocyanin complex was emulsified in full Freund's adjuvant and injected subcutaneously into BALB/c mice.

Sera were spun at 500 x g for 5 min, diluted in 5% milk in PBS/Brij, and analyzed by Western blotting against recombinant MUC1/ZD.

Hybridoma Formation—Hybridomas were prepared by fusion of immune splenocytes with the NS-O myeloma cell line, kindly provided by C. Milstein (35), and screened by an ELISA. Hybridoma supernatants were tested against wells of ELISA plates coated with either GST-MUC1/ZD or GST-MUC1/N-terminal fusion proteins. The MUC1/N-terminal protein extends from MUC1 residue Ser24 to Pro128. Bound antibodies were detected by incubation with alkaline phosphatase-conjugated anti-mouse antibodies.

Protein A-Sepharose Purification of ZUM12D8/15 and ZUM7E7 Anti-MUC1/ZD Monoclonal Antibodies—ZUM12D8/15 and ZUM7E7 anti-MUC1/ZD (anti-MUC1/ZD) and anti-MUC1-tandem-repeat array H23 are all Ig{gamma}1 and were purified using protein A-Sepharose. Fluorescent labeling and biotin conjugation of antibodies was done as described (Molecular Probes and Pierce, respectively).

ELISA for Determining MUC1/ZD Protein Concentration—ELISA immunoassay plates (Costar) were coated with purified ZUM7E7 followed by washing with PBS-Tween 20 (0.05%) and blocking with PBS-Tween 20 + 5% skimmed milk (Blotto). Doubling dilutions of either recombinant MUC1/ZD or sample to be analyzed were diluted with Blotto and applied to wells. Following incubation, samples were removed and wells were washed four times with PBS-Tween 20. Biotinylated ZUM12D8 then was added (5 µg/ml incubated for 30 min) followed by detection with horseradish peroxidase-conjugated streptavidin.

SDS-PAGE—SDS-polyacrylamide gel for protein separation was performed as described previously (36).

Two-dimensional SDS-PAGE Diagonal Gel Electrophoresis—Samples analyzed by SDS-PAGE were resolved in two dimensions following a modification of a described previously procedure (37). The first dimension was performed under non-reducing conditions. For the second dimension, the desired lanes were excised from the first gel, incubated with SDS loading buffer with or without 100 mM dithiothreitol, and reloaded horizontally on a second gel.

Transfer of Proteins to Nitrocellulose Membranes and Immunoblotting Analysis—Proteins, separated on SDS-PAGE, were electrotransferred at 0.5 A for 2 h to a nitrocellulose filter paper in transfer buffer according to Gershoni (38). Blots were blocked in 5% skimmed milk followed by incubation with primary antibody. Bound primary antibody was detected with a secondary anti-mouse antibody conjugated to horseradish peroxidase followed by enhanced chemiluminescence.

Purification of Proteins from SDS-PAGE Gels and Protein Iodination—Proteins were extracted from the acrylamide gel as described previously. The 60–65-kDa regions were excised from the gel, and proteins were eluted by shaking in elution buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% SDS, 0.1 mM EDTA, 5 mM dithiothreitol) followed by acetone precipitation and renaturation of the precipitated proteins. Purified proteins were incubated with 125I-conjugated Bolton-Hunter reagent (200 mCi) for 15 min on ice. The reaction was terminated by the addition of glycine, and the solution was passed through a Sephadex G-50 column to remove free iodine.

Immunoprecipitation—Immobilized antibody complexes were prepared by incubating protein A (PAA Repligen) with 5–10 µg of antibody followed by incubation at 4 °C. Incubation of cell lysate with a nonrelevant antibody-PAA complex yielded precleared lysates that were then subjected to immunoprecipitation with the specific antibody-PAA complex. Following washing, the immunoprecipitated proteins were analyzed on SDS-PAGE.

Expression of MUC1/ZD-Green Fluorescent Protein (GFP) Fusion Protein in Eucaryotic Cells—MUC/ZD cDNA was inserted into the pEGFP-N3 vector (Clontech) harboring the cytomegalovirus IE promoter. In this expression system, the open reading frame codes for a MUC/ZD-enhanced GFP fusion protein in which MUC/ZD is located N-terminal to GFP. Plasmids harboring the MUC/ZD-enhanced GFP-coding sequence were used for transient transfection of HEK293 cells.

Immunohistochemical Staining of Frozen Sections—Thawed frozen sections were fixed in acetone and blocked with PBS containing 2.5% skimmed milk and 50% normal mouse serum. The slides then were incubated with the fluorescently labeled antibodies followed by washing, air-drying, mounting, and analysis by fluorescent or confocal laser microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A Tandem-Repeat Array-lacking MUC1 mRNA with a +1 Frameshifted 3' End—The splicing event that generates MUC1/Y (Fig. 1A2) spliced out the central tandem-repeat array and its immediate flanking sequences. RT-PCR identified an additional alternatively spliced tandem-repeat array-lacking MUC1 mRNA termed here as MUC1/ZD (Fig. 1A3). The MUC1/ZD mRNA isoform utilized a splice donor site identical to that used by MUC1/Y and could have generated a protein at its N terminus identical to the transmembrane MUC1/Y protein, including the signal peptide. As a consequence of alternate splice acceptor usage and in contrast to MUC1/Y, the MUC1/ZD reading frame distal to the splice acceptor site was +1 frameshifted (Fig. 1, A3 and B). MUC1/ZD mRNA 3' to the MUC1/ZD termination codon was identical to that of the MUC1/Y and MUC1/REP sequences. The MUC1/ZD C-terminal unique region spanned 43 amino acids and was completely different from any other known MUC1 protein. In contrast to the transmembrane MUC1/REP and MUC1/Y proteins, MUC1/ZD did not contain a transmembrane domain.

MUC1/ZD Protein Expression in Western blots—Western blots of tissue lysates prepared from human tumor samples were probed with antibodies generated against bacterial recombinant GST-MUC1/ZD protein. The expected size of the MUC1/ZD protein (73 amino acids) predicted that it should migrate in the region just above the 6-kDa marker protein. Immunoreactive bands migrating with this molecular weight were observed in three of eight tissue samples analyzed (Fig. 2A, lanes 1, 4, and 7; samples 1 and 4 are from colon tumors, and sample 7 is from breast tumor). We do not know whether higher MUC1/ZD expression in colon tumor tissue, as compared with breast tumor tissue, is a general finding, and this will require a larger study beyond the scope of the present work. Competing MUC1/ZD protein abrogated immunoreactivity (Fig. 2B, GST-MUC1/ZD). The addition of the peptide TTTKSCRETFLK, an immunogenic MUC1/ZD epitope, in a Western blot analysis also competed out immunoreactivity (Fig. 2B, peptide). Two splice variants, AY327597 [GenBank] and AY327598 [GenBank] , that are similar yet different from MUC1/ZD appear in the data base. These variants have the potential to code (following signal peptide cleavage) for ~11.5-kDa (105 amino acids) and 15-kDa (135 amino acids) proteins. These variants do not contain the MUC1/ZD-specific C-terminal octamer sequence GQDLWWYN and instead continue (after the MUC1/ZD sequence WASPILSS (see Fig. 1) for an additional 40 or 70 amino acids (GenBankTM accession numbers AY327597 [GenBank] and AY327598 [GenBank] , respectively)). The molecular mass of the MUC1/ZD protein observed here (~7–8 kDa) correlates well with the expected size of the MUC1/ZD protein and is considerably less than the expected masses of these two variants.



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  		FIG. 2.
Immunoblotting of MUC1/ZD protein in tissue lysates. Protein lysates from human carcinoma tissues were resolved by 12.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, probed with either affinity-purified rabbit polyclonal anti-MUC1/ZD antibodies (top panel in A and B) or affinity-purified antibodies directed against MUC1 cytoplasmic domain (lower panel in A). Detection was achieved by enzyme-linked goat anti-rabbit antibodies followed by ECL as described under "Experimental Procedures." B, to determine anti-MUC1/ZD specificity, either GST-MUC1/ZD recombinant protein (GST-MUC1/ZD) or a 12-mer peptide, TTTKSCRETFLK, derived from MUC1/ZD-unique region (peptide) were added at 100-fold molar excess to the anti-MUC1/ZD antibody preparation. Human carcinoma tissues were derived either from colorectal lesions (A, lanes 1 and 4, and B, lanes 2 and 3) or from breast tumors (A, lanes 2, 3, 5, 6, and 7, and B, lane 1).

 
Reprobing of the blot with anti-MUC1 cytoplasmic domain antibodies demonstrates immunoreactive bands migrating between 25 and 30 kDa (Fig. 2A, lower panel, lanes 2 and 5, with lesser amounts observed in lanes 3 and 7). These bands represent the MUC1 cytoplasmic domain-containing cleavage products, reflecting MUC1/REP expression. The expression of the MUC1/ZD protein did not correspond with the expression of the MUC1/REP protein, and only one tissue sample (Fig. 2, lane 7) showed expression of both the MUC1/ZD and MUC1/REP proteins.

MUC1/ZD Protein Expression by Immunohistochemical Staining—Immunohistochemical analysis substantiated MUC1/ZD protein expression in human tissues and demonstrated the expression of the MUC1/ZD protein in breast tumor tissue (Fig. 3A). The addition of the immunizing MUC1/ZD protein abrogated immunohistochemical reactivity (Fig. 3B), supporting specificity of antibody staining. In line with these findings, the addition of GST protein itself had no effect on immunoreactivity (Fig. 3C). Taken together, the Western blot and immunohistochemical analyses demonstrated MUC1/ZD expression at the protein level in human tissues.



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  		FIG. 3.
Immunohistochemical analysis of breast tumor tissue with anti-MUC1/ZD antibodies. Paraffin-embedded sections from human breast cancer tissue were immunostained with affinity-purified anti-MUC1/ZD polyclonal antibodies (A). 100-fold molar access of either recombinant bacterial MUC1/ZD (B) or recombinant MUC1 N-terminal domain (C) was added to the antibody solution prior to the incubation with the tissue sections. Panels A–C, 200-fold magnification. Panels A'–C', 1000-fold magnification. The brown staining in panels A and C represents expression of the MUC1/ZD protein.

 
The MUC1/ZD Protein Exists as a Disulfide-linked Oligomer—The unique MUC1/ZD 43 amino acid C-terminal domain contained three cysteine residues (Fig. 1B, highlighted amino acid residues) that may form intramolecular and/or intermolecular disulfide bonds. To test for this possibility, the lysate of a tissue known to express the MUC1/ZD protein was analyzed under both reducing and non-reducing conditions and then was subjected to a Western blot analysis. All of the samples for these analyses were prepared with buffer containing iodoacetamide. Under reducing conditions, the tissue lysate displayed an immunoreactive band migrating just above the 6-kDa marker protein (Fig. 4A, lane 1). Under non-reducing conditions, the low molecular mass species (6–7 kDa) was no longer seen (Fig. 4A, lane 1'). In its place, a distinct immunoreactive band migrating at ~64 kDa was observed. To detect the small MUC1/ZD monomeric protein, Western blots described above (and below) (Fig. 4, C–E) were done with high percentage (12.5–15%) acrylamide gels. Although advantageous for the resolution of small proteins, considerable stacking and piling up of higher molecular weight proteins occurs in such gels. In Western blots, this could result in artifactual bands representing proteins that are nonspecifically immunoreactive. To address this possibility, MUC1/ZD-positive and negative samples (as determined by Western blots of reduced samples) were reassessed under non-reducing conditions with a lower percentage (10%) acrylamide gel. Immunoblotting with anti-MUC1/ZD antibodies demonstrated a strong 64-kDa immunoreactive band in the MUC1/ZD-positive sample (Fig. 4B, lane 1). No immunoreactivity was seen with the MUC1/ZD-negative sample (Fig. 4B, lane 2). No significant additional nonspecific immunoreactive bands were observed. These data indicated that the 64-kDa band represents the MUC1/ZD protein. The additional bands appearing in high percentage acrylamide gels probably represented proteins that, because of protein stacking and piling up, reacted nonspecifically. This conclusion is supported by immunoprecipitation experiments described below.



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  		FIG. 4.
The MUC1/ZD protein exists as a disulfide-linked oligomer. A, protein lysate from a human carcinoma tissue that expresses MUC1/ZD was resolved on 12.5% SDS-PAGE either under reducing (lane 1) or non-reducing (lane 2) conditions and analyzed by Western blotting using affinity-purified anti-MUC1/ZD polyclonal antibodies. B, protein lysates from human carcinoma tissues that express or do not express MUC1/ZD (lanes 1 and 2, respectively) were resolved on 10% SDS-PAGE gels under non-reducing conditions and analyzed by Western blotting using affinity-purified anti-MUC1/ZD polyclonal antibodies. C, human carcinoma tissue that expresses MUC1/ZD and a non-expressing human carcinoma tissue were resolved on 10% SDS-PAGE under non-reducing conditions. Subsequently, the 64-kDa region was excised from the gel, the proteins were eluted, and 125I-labeled and equal amounts of radiolabeled proteins from the two samples were immunoprecipitated with affinity-purified anti-MUC1/ZD polyclonal antibodies. Labeled precipitating proteins from MUC1/ZD expressing and non-expressing tissues (lanes 1 and 2, respectively) were analyzed on 12.5% SDS-PAGE under non-reducing conditions. D, the labeled precipitating proteins from MUC1/ZD-expressing tissue (as in C) were analyzed on 12.5% SDS-PAGE under non-reducing and partially reducing conditions (lanes 1 and 1', respectively). E, lysate from human carcinoma tissue that expresses MUC1/ZD was analyzed by diagonal gel electrophoresis. The first dimension was resolved under non-reducing conditions (1'(-Red.)), whereas the second dimension was performed either under non-reducing conditions (E1, 2'(-Red.)) or under reducing conditions (E2, 2'(+Red.)). Subsequently, the gel was analyzed by Western blotting using affinity-purified anti-MUC1/ZD polyclonal antibodies. The open arrow (in panel E1) indicates the non-reduced MUC1/ZD complex. The black arrow (in panel E2) indicates the reduced MUC1/ZD monomer. The dashed circle (in panel E2) indicates the expected location of the non-reduced MUC1/ZD complex.

 
Proteins extracted from two tumor tissue lysates, one considered a known MUC1/ZD-positive sample and the other a MUC1/ZD-negative sample, were analyzed under identical non-reducing gel electrophoretic conditions. Proteins migrating in the 64-kDa region were eluted from the gel and radioiodinated, and equivalent levels of radioactivity (from both samples) were immunoprecipitated using anti-MUC1/ZD antibodies. Immunoprecipitation revealed a 64-kDa band only in the MUC1/ZD-expressing sample (Fig. 4C, compare lanes 1 and 2). Partial reduction of the immunoprecipitated MUC1/ZD 64-kDa protein complex resulted in a ladder of radioactively labeled bands (Fig. 4D, lane 1') that probably reflected MUC1/ZD oligomers containing different numbers of monomeric subunits that were disulfide-bonded to each other. Regarding their actual molecular mass, disulfide-bonded proteins often migrate anomalously on SDS-PAGE. This could explain why observed distances between labeled bands (Fig. 4D, lane 1') do not in all cases correlate with the expected 7-kDa size of a MUC1/ZD monomer. Alternatively, the MUC1/ZD complex may comprise additional non-MUC1/ZD proteins.

The 64-kDa MUC1/ZD protein was analyzed next by gel electrophoresis performed in two dimensions. Two identical lanes were analyzed in the first dimension under non-reducing conditions. Following electrophoresis, one lane was taken and placed horizontally on a new gel and analyzed in the second dimension, once again under non-reducing conditions. The second lane was soaked in reducing sample buffer and analyzed on a new gel under reducing conditions. The two gels were blotted and probed with anti-MUC1/ZD. If the 64-kDa MUC1/ZD protein is not disulfide-bonded, it should appear in both Western blots at a position located on the diagonal of the two-dimensional blot. On the other hand, if the 64-kDa MUC1/ZD protein is a disulfide-bonded oligomer of a 6–7-kDa MUC1/ZD monomer, it should appear on the diagonal only in the non-reduced second gel. In the reduced second dimension gel, it would appear at a location below the diagonal and correspond to migration of a 6–7-kDa protein. This is indeed the case (Fig. 4, E1 and E2, compare spots designated by arrowheads).

Oligomeric MUC1/ZD complexes were also observed in lysates prepared from cell transfectants expressing a MUC1/ZD-GFP fusion protein (Fig. 5). It should be kept in mind that the MUC1/ZD-GFP protein is a synthetic recombinant fusion protein different from wild-type MUC1/ZD. As such, it may not faithfully replicate all of the properties of native MUC1/ZD. Probing with anti-GFP antibodies of reduced samples demonstrates a discrete MUC1/ZD-GFP band (Fig. 5A, left panel, lane 2). A ladder of immunoreactive MUC1/ZD-GFP bands was observed with the partially reduced sample (compare Fig. 5A, lanes 2 in the left and right panels). Control cells transfected with a vector coding for GFP protein alone displayed a discrete band irrespective of sample reduction status (compare Fig. 5A, lane 1 in the left and right panels). Immunoreactive bands reactive with anti-GFP antibodies (commercially acquired) migrating with molecular masses above 60 kDa were also seen in non-transfected cells and were probably nonspecific bands (Fig. 5A, left panel, lanes 1–3).



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  		FIG. 5.
MUC1/ZD forms oligomeric complexes when expressed in 293 cells. A, protein lysates from 293 cells (lane 3, 293) or from 293 cells transfected either with the MUC1/ZD-GFP cDNA (lane 2, Z-GFP) or with the GFP cDNA alone (lane 1, GFP) were separated on 11% SDS-PAGE under reducing (A) or partially reducing (B) conditions. Samples were analyzed by Western blot using anti-GFP antibodies. The high molecular weight bands in panel A (left panel) are not specific. B, 293 cells (lane 1, 293) or 293 cells transfected either with the GFP cDNA (lane 2, GFP) or with the MUC1/ZD-GFP cDNA alone (lane 3, Z-GFP) were metabolically labeled with [35S]methionine/cysteine, lysed, and immunoprecipitated with anti-MUC1/ZD antibodies. Immunocomplexes were separated on 11% SDS-PAGE under reducing (B, left panel) or partially reducing (B, right panel) conditions. The arrows to the right indicate the MUC1/ZD-GFP oligomeric complexes. The gel was amplified, dried, and exposed to film.

 
Proteins from cell transfectants expressing MUC1/ZD-GFP protein were radioactively labeled and immunoprecipitated with anti-MUC1/ZD antibodies. A ladder of labeled bands was observed under partially reducing conditions (Fig. 5B, right panel, lane 3). An analysis under reducing conditions clearly showed one labeled band corresponding to the monomeric MUC1/ZD-GFP protein. No additional bands were seen (Fig. 5B, left panel, lane 3). No labeled bands were observed following anti-MUC1/ZD immunoprecipitations of labeled lysates from 293 or 293 cells transfected with GFP alone (Fig. 5B, lanes 1 and 2). An analysis of secreted protein products showed a ladder of MUC1/ZD-GFP bands similar to those seen in Fig. 5B (data not shown). Rather than a discrete oligomeric MUC1/ZD-GFP protein band as seen with native MUC1/ZD protein (Fig. 4, A, C, D, and E), non-reduced gels of the recombinant MUC1/ZD-GFP fusion protein demonstrated patterns similar to those seen in the partially reduced gels (data not shown). In contrast to native MUC1/ZD, MUC1/ZD-GFP is a synthetic recombinant fusion protein and may not adopt the wild-type MUC1/ZD conformation required for the formation of the optimal disulfide-bonded 64-kDa MUC1/ZD protein (Fig. 4). This may explain the disulfide-bonded oligomer ladders observed here (Fig. 5) for the MUC1/ZD-GFP protein.

Taken together, all of the above results support the notion that MUC1/ZD exists as an oligomeric complex composed of MUC1/ZD protein subunits that are disulfide-bonded to each other.

Generation of Anti-MUC1/ZD Monoclonal Antibodies—Mice were immunized with a GST-MUC1/ZD fusion protein, and hybridoma supernatants were screened for immunoreactivity against both the GST-MUC1/ZD fusion protein and, in this case, non-relevant GST-MUC1/N-terminal protein (see "Experimental Procedures"). Two particularly reactive clones, ZUM12D8 and ZUM7E7, were obtained and chosen for further study. These hybridomas secreted immunoglobulins (both Ig{gamma}1 subtypes) that reacted only with the MUC1/ZD protein (Fig. 6A). They were non-reactive toward the MUC1/Y protein (Fig. 6B) and did not react with the N-terminal portion of MUC1 protein (Fig. 6C) and thus were specific for MUC1/ZD. The ZUM7E7 and ZUM12D8 monoclonal antibodies recognized the reduced bacterial recombinant MUC1/ZD protein (Fig. 6D, lanes 1 and 3).



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  		FIG. 6.
Specificity of monoclonal antibodies ZUM12D8 and ZUM7E7 for MUC1/ZD protein. A–C, 20 ng/lane MUC1/ZD protein (panel A), DHFR-MUC1/Yex (the extracellular domain of MUC1/Y fused to the C terminus of the non-relevant bacterial DHFR protein, panel B), and GST-N-terminal MUC1/REP (the N-terminal sequences of MUC1/REP extending from the site of signal peptide cleavage to the tandem-repeat array fused to the C terminus of GST, panel C) were resolved on 12% SDS-PAGE, blotted, and probed with MUC1/ZD-specific monoclonal antibody ZUM12D8 (lanes 1), MUC1/ZD-specific monoclonal antibody ZUM7E7 (lanes 2), and MUC1/Y-specific monoclonal antibody BOS6E6 (lanes 3). Bound mouse monoclonal antibodies were detected by enzyme-linked anti-mouse antibodies followed by ECL as described under "Experimental Procedures." D, 20 ng/lane bacterial recombinant MUC1/ZD protein were resolved under reducing conditions on 12% SDS-PAGE, blotted, and probed with MUC1/ZD-specific monoclonal antibodies ZUM7E7 and ZUM12D8 as indicated. Immunoblotting was performed with or without the addition of peptide (pep.) TTTKSCRETFLK as indicated.

 
The peptide TTTKSCRETFLK present in the C-terminal 43 MUC1/ZD-unique amino acids was highly hydrophilic, suggesting that it may be an immunogenic site. The addition of this peptide reduced ZUM7E7 immunoreactivity (Fig. 6D, compare lanes 1 and 2). It was without any effect on ZUM12D8 (Fig. 6D, compare lanes 3 and 4). This finding suggested that the MUC1/ZD-specific monoclonal antibodies recognize distinct epitopes, supporting the development of a sandwich ELISA for MUC1/ZD protein detection.

An ELISA for Detecting MUC1/ZD Protein—ELISA formats were established in which capture antibody consisted of ZUM7E7 and detecting antibody comprised biotinylated ZUM12D8. This assay detected MUC1/ZD down to a protein concentration of ~50 pg/ml, and no cross-reactivity with MUC1/Y or any other MUC1 protein was observed.

Using this assay, we screened sera from four normal individuals as well as sera from ten breast cancer patients. No MUC1/ZD protein could be detected. Effusions obtained by aspirating fluids from the sites of breast lumpectomies were examined next for the presence of MUC1/ZD. Eight separate effusion samples originating from a total of seven patients were investigated. MUC1/ZD protein was found in the effusions of only one patient at levels corresponding to ~2 ng/ml (40-fold higher than assay detection limit). Two separate samples aspirated at different time intervals (3 and 7 days) following lumpectomy were obtained from this same patient. Both samples were positive for the MUC1/ZD protein with slightly higher levels present in the effusion sample obtained at the earlier post-lumpectomy time point.

Immunohistochemical Analysis of MUC1/ZD Expression with Monoclonal Antibodies—Preliminary immunohistochemical studies demonstrated that ZUM12D8 could detect MUC1/ZD protein in tissue sections. The best immunoreactivity was obtained with acetone-fixed frozen tissue sections. Because substantial expression of the MUC1 gene in skin and in sebaceous gland tumors has been recently documented (39), we analyzed MUC1 gene expression in frozen skin tissue sections. Purified ZUM12D8 anti-MUC1/ZD monoclonal antibody was green fluorescently labeled and direct immunofluorescence on frozen skin tissues assessed. MUC1/ZD immunoreactivity was readily detected (see Fig. 7, A, D, and G, and compare with hematoxylin-stained consecutive sections in C, F, and I). Anti-MUC1/ZD immunoreactivity was limited to epithelial cells comprising sebaceous glands, hair follicles, and epithelial cell layers forming skin surface. Essentially all of the immunoreactivity was competed out by adding immunizing MUC1/ZD protein (compare panels A, D, and G with panels B, E, and H). Immunoreactivity was not observed in either the fibroblasts embedded within the connective tissue or in the connective tissue itself.



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  		FIG. 7.
Immunohistochemistry of skin tissue sections with green fluorescently labeled anti-MUC1/ZD-specific monoclonal antibody ZUM12D8. Frozen sections of skin tissue were stained with green fluorescently labeled (Alexa Fluor 488, Molecular Probes) anti-MUC1/ZD monoclonal antibody ZUM12D8 as described under "Experimental Procedures" without any competitor (A), with the addition of competing excess MUC1/ZD protein to labeled antibody reagent (B), or eosin/hematoxlyn-stained (C). Panels D, E, and F and panels G, H, and I represent different skin tissue sections subjected to same treatments as for A, B, and C. Note intense staining in A, D, and G of epithelial cells forming sebaceous glands and surface skin epithelial cells. No staining is observed with fibroblasts or with any connective tissue components. All of the immunoreactivity was competed out by the addition of competing soluble MUC1/ZD protein (panels B, E, and H).

 
To corroborate ZUM12D8 staining, the second anti-MUC1/ZD monoclonal antibody ZUM7E7 was red fluorescently labeled for immunostaining frozen skin sections. Immunoreactivity was similar to that obtained with green fluorescently labeled ZUM12D8 (Fig. 8A). Simultaneous immunostaining with red-labeled ZUM7E7 and green-labeled ZUM12D8 showed direct overlap of the green and red immunostaining (Fig. 8A4). The MUC1/ZD-specific peptide, TTTKSCRETFLK, previously determined to be part of the ZUM7E7 epitope (Fig. 6E, lanes 1 and 2) substantially reduced ZUM7E7 immunoreactivity (Fig. 8B1). The addition of a control peptide located within the MUC1 30 amino acids downstream to signal peptide cleavage was of no effect.



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  		FIG. 8.
Immunohistochemistry of skin tissue sections with green fluorescently labeled ZUM12D8 and red-labeled ZUM7E7 anti-MUC1/ZD-specific monoclonal antibodies. Frozen skin tissue sections were double-stained with green fluorescently labeled (Alexa Fluor 488) ZUM12D8 and red fluorescently labeled (Alexa Fluor 546) ZUM7E7 as described under "Experimental Procedures." The stained section was analyzed by confocal laser microscopy (200-fold magnifications) calibrated for the following: 1, red fluorescence representing detection of MUC1/ZD by ZUM7E7; 2, green fluorescence representing detection of MUC1/ZD by ZUM12D8; 3, Nomarski optics images of the same sections, and 4, a merged overlay of the two signals (1 and 2). Immunostaining was performed either with the addition of a nonrelevant peptide (panels A) or with the MUC1/ZD peptide, TTTKSCRETFLK (panels B).

 
MUC1/ZD expression was compared next with that of the classical transmembrane tandem-repeat array-containing protein, MUC1/REP. A double-staining procedure with red fluorescently labeled anti-MUC1 tandem-repeat array H23 antibodies and green fluorescently labeled anti-MUC1/ZD ZUM12D8 antibodies demonstrated the expression of both MUC1 proteins in epithelial cells comprising the sebaceous gland (Fig. 9), the cellular architecture of which was similar to that shown in Fig. 7F. Some labeled cells displayed the expression of only one of the two isoforms (compare Fig. 9, A and B). Epithelial cells located on the margin of the sebaceous gland primarily expressed the MUC1/ZD protein (Fig. 9A, green fluorescence), whereas cells located at the gland interior displayed red fluorescence signifying MUC1/REP expression (Fig. 9B). A computer-generated overlay of the two fluorescent signals demonstrated that some cells expressed almost equal levels of MUC1/ZD and MUC1/REP protein isoforms (Fig. 9C, yellow fluorescence). Higher magnification analyses of those cells expressing both MUC1 isoforms indicated that MUC1/REP protein was located almost exclusively on the cell surface, as expected of a membrane-bound protein (Fig. 9E, red fluorescence). In contrast, MUC1/ZD protein was seen both on the cell surface as well as localizing to interstitial spaces (Fig. 9D, green fluorescence). A computer-generated merge confirmed this assessment (Fig. 9F, yellow designates regions expressing both MUC1/REP and MUC1/ZD, whereas green indicates MUC1/ZD expression alone).



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  		FIG. 9.
Immunohistochemical double staining of a skin tissue sample with green fluorescently labeled anti-MUC1/ZD-specific monoclonal antibody ZUM12D8 and red fluorescently labeled anti-MUC1 tandem-repeat-specific monoclonal antibody H23. A skin tissue sample was immunostained as described under "Experimental Procedures" and in Fig. 6 with a solution containing green fluorescently labeled (Alexa Fluor 488) anti-MUC1/ZD monoclonal antibody ZUM12D8 together with red fluorescently labeled (Alexa Fluor 546) anti-MUC1/REP tandem-repeat monoclonal antibody H23. The stained section was analyzed by confocal laser microscopy calibrated for green fluorescence representing detection of MUC1/ZD (A), red fluorescence representing detection of MUC1/REP (B), and a merged overlay of the two signals (C). Panels A–C are 200-fold magnifications. Panels D–F are as A–C 1000-fold magnifications.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We describe a novel MUC1 protein, MUC1/ZD, which is derived from the MUC1 gene. MUC1/ZD does not contain the hallmark tandem-repeat array domain distinctive of mucin proteins, and the complete C-terminal segment is a novel peptide sequence absent from all of the other known MUC1 protein isoforms. The generation of MUC1/ZD results from an alternative splicing event, which deletes the central tandem-repeat array and effects a +1 change in the reading frame immediately downstream to the splice acceptor site.

The expression of the novel MUC1/ZD protein has been demonstrated using affinity-purified polyclonal antibodies and MUC1/ZD-monospecific monoclonal antibodies by Western blotting, immunohistochemistry, immunoprecipitation, and ELISAs. The polyclonal and monoclonal antibody reagents used here recognize epitopes located in the frameshifted MUC1/ZD C-terminal sequence, a region predicted to be present in the alternative splice forms, AY327597 [GenBank] and AY327598 [GenBank] . The MUC1/ZD-specific C-terminal octamer sequence, GQDLWWYN, is not present in these variants, and instead they continue (after the MUC1/ZD sequence WASPILSS, see Fig. 1) for an additional 40 or 70 amino acids (AY327597 [GenBank] and AY327598 [GenBank] , respectively). In immunoblots probed with anti-MUC1/ZD polyclonal antibodies, these larger putative proteins would not be expected to migrate with a molecular mass of 7–8 kDa as observed here for MUC1/ZD. However, it is possible that the anti-MUC1/ZD monoclonal antibody reagents are recognizing, in addition to MUC1/ZD, proteins derived from the putative AY327597 [GenBank] and AY327598 [GenBank] mRNAs. This caveat notwithstanding, it is clear nonetheless from the work presented here that human cells express novel frameshifted MUC1 proteins. As such, this report is the first demonstration of expression in human cells of frameshifted MUC1 proteins.

MUC1/ZD expression is observed both in cancer tissue and in epithelial cells comprising the epithelial skin layers and sebaceous glands. Interestingly, its expression did not always correlate with that of the MUC1/REP protein. This observation suggests that alternative splicing determines which MUC1 mRNA species is expressed and that, depending on its specific needs at any particular time, the cell may require expression of a specific combination of the MUC1 proteins.

The MUC1/ZD protein presents as an oligomeric protein complex. This oligomer is probably composed of monomeric MUC1/ZD subunits that interact with each other by disulfide bonds (Fig. 4, A and B). Indeed MUC1/ZD comprises three cysteine residues containing reactive sulfhydryl groups that may form covalent disulfide intermolecular disulfide bonds. Secreted ligands that act as disulfide-bonded dimers may exist as either homodimers or heterodimers (40, 41). Oligomers consisting of more than two subunits are also documented, although they are less common. The MUC1/ZD oligomer is obviously not a dimeric protein, and its molecular size suggests that it comprises approximately 8–10 MUC1/ZD monomeric subunits. Although immunoprecipitation studies of radioactively labeled cells expressing a MUC1/ZD fusion protein do not show any evidence of other proteins(s) present in the MUC1/ZD complex, it is still possible that the MUC1/ZD oligomer comprises heterologous proteins covalently bonded to MUC1/ZD.

The function of the expressed frameshifted MUC1/ZD protein is not clear. It is possible that, although being expressed, the frameshifted MUC1/ZD protein (and perhaps similar MUC1-frameshifted proteins) may have no biological function. However, a tblastn search did reveal limited similarity with a group of proteins that function in the innate immune system (42–45). The best fit is seen between MUC1/ZD and CD14, a protein that binds bacterial surface components (46). Although by no means extensive, this similarity is noteworthy in that the homologous region in MUC1/ZD comprises the cysteine residues. Additional studies will be required to establish whether MUC1/ZD or similar frameshifted proteins perform functions related to innate immunity.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY466157 [GenBank] (locus) and AAR28764 [GenBank] 1 (protein).

* This work was supported by U. S. Army Medical Research Grant DAMD 17-00-1-0451 (to D. H. W.). 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. Back

§ Both authors contributed equally to this work. Back

§§ To whom correspondence should be addressed. Tel.: 972-3-6407425; Fax: 972-3-6422046; E-mail: danielhw{at}post.tau.ac.il.

1 The abbreviations used are: RT, reverse transcription; GFP, green fluorescent protein; GST, glutathione S-transferase; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. Back



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F. Levitin, O. Stern, M. Weiss, C. Gil-Henn, R. Ziv, Z. Prokocimer, N. I. Smorodinsky, D. B. Rubinstein, and D. H. Wreschner
The MUC1 SEA Module Is a Self-cleaving Domain
J. Biol. Chem., September 30, 2005; 280(39): 33374 - 33386.
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