Human Molecular Genetics, 2000, Vol. 9, No. 14 2167-2173
© 2000 Oxford University Press
Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart
MRIC Biochemistry Group, PP18, North East Wales Institute, Mold Road, Wrexham LL11 2AW, UK
Received 11 May 2000; Accepted 28 June 2000.
DDBJ/EMBL/GenBank accession no. AF250871.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Myotonic dystrophy (DM) is a multisystemic disorder caused by an inherited CTG repeat expansion which affects three genes encoding the DM protein kinase (DMPK), a homeobox protein Six5 and a protein containing WD repeats. Using a panel of 16 monoclonal antibodies against several different DMPK epitopes we detected DMPK, as a single protein of
80 kDa, only in skeletal muscle, cardiac muscle and, to a lesser extent, smooth muscle. Many earlier reports of DMPK with different sizes and tissue distributions appear to be due to antibody cross-reactions with more abundant proteins. One such antibody, MANDM1, was used to isolate two related protein kinases, MRCK
and ß, from a human brain cDNA library and the shared epitope was located at the catalytic site of DMPK using a phage-displayed random peptide library. The peptide library also identified an epitope shared between DMPK and a 55 kDa muscle-specific protein. The results suggest that effects of the repeat expansion on the DMPK gene may be responsible for muscle and heart features of DM, whereas clinical changes in other tissues may be due to effects on the other two genes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Myotonic dystrophy (DM) is caused by an unstable CTG repeat sequence in the 3'-untranslated region of a protein kinase (DMPK) gene on chromosome 19 (1–3). The CTG repeat increases from 5–30 in the normal population to 50–1000+ in DM patients and the increase is correlated with disease severity (4). Nuclear retention of DMPK transcripts with expanded CTG repeats has been demonstrated (5) and decreased DMPK poly(A)+ mRNA levels have been reported in DM tissues (6,7). The expanded CTG repeat at the DM locus is also within the 5' end of the Six5 gene predicted to encode a homeobox protein (8) and a third gene, DMWD (gene 59, or DMR-N9 in the mouse), which lies 5' to DMPK, is also affected by CTG expansions (9). Effects on expression of non-DMPK proteins could therefore be responsible for some, if not all, of the clinical features of DM. DM affects many different tissues, including muscle (myotonia and progressive weakness), brain (mental retardation), eyes (cataracts), testis (atrophy) and heart (conduction defects) (10). It is important, therefore, to establish the normal tissue distribution of all three proteins produced from the DM locus.
The human DMPK cDNA has 15 exons and predicts a protein of 70 kDa, although there may be variation in the size and sequence of the first exon (1–3,11,12). DMPK is a serine/threonine protein kinase in which the catalytic domain (43 kDa) is followed by a coiled-coil domain (12 kDa) and a hydrophobic C-terminal domain. Alternative splicing has been observed in a short VSGGG sequence between domains and in the C-terminal domain (13). Characterization of DMPK protein using antibodies has proven to be very difficult and is still a source of considerable confusion. This may be largely due to antibody cross-reaction, to which DMPK antibodies seem to be particularly vulnerable. Anti-peptide antisera against the catalytic domain have recognized proteins of 52–55 kDa in skeletal muscle (14,15), 42 and 60 kDa in brain and heart (14,15) or 53 kDa in heart and skeletal muscle (16). Several antisera prepared by Timchenko et al. (17) did recognize a protein of the expected size (72 kDa) in fibroblasts. A monoclonal antibody produced using recombinant catalytic and coil domains as immunogen recognized a 64 kDa protein in muscle and a 79 kDa protein in brain (18). However, variable results have also been obtained with antibodies against the C-terminal domain, which is not known to share sequences with any other protein. Polyclonal antisera raised against the whole C-terminal domain recognized proteins of 71–74 and 80–85 kDa in skeletal and cardiac muscle (19–21). However, a similar study produced antiserum recognizing proteins of 85 and 54 kDa (22) and antisera against C-terminal peptides recognized proteins of 70–72 and 50–55 kDa in skeletal muscle (23,24). Finally, a 45 kDa protein was detected in brain using C-terminal antibodies (21).
The correct approach to this problem is to prepare a panel of monoclonal antibodies (mAbs) which recognize several different epitopes on DMPK. Although mAbs, like antisera, often display cross-reactions with other proteins, these cross-reactions tend to be very specific for each epitope. Any protein on western blots which contains most, if not all, of the epitopes is likely to be authentic DMPK, whereas proteins sharing only one epitope with DMPK are likely cross-reactions. Our initial results with a panel of 12 mAbs against the catalytic or coil domains suggested that DMPK was expressed as 80 and 72 kDa proteins, the 80 kDa protein being present mainly in skeletal and cardiac muscle whereas the 72 kDa protein is widely expressed (25). We now show, however, that only the 80 kDa protein is authentic DMPK and that it is detectable only in skeletal muscle, in cardiac muscle and, at lower levels, in smooth muscle. Cross-reacting proteins of 190 and 72 kDa recognized by mAb MANDM1 appear to be isoforms of myotonic dystrophy-related cdc42-binding kinase (MRCK) (26–28).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A panel of 12 mAbs, prepared using a DMPK fragment containing the catalytic and coil domains, has been described previously (25). Seven mAbs recognized the catalytic domain and five recognized the coil domain. A further four mAbs, MANDM13–16, were prepared using recombinant coil plus C-terminal domains and all four of these mapped to the coil domain (Table 1).
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Figure 1a shows that the only protein recognized by nearly all mAbs on a western blot of human skeletal muscle is a single band of
80 kDa. Since these mAbs recognize several different epitopes and they all recognize the DMPK recombinant fusion protein (data not shown), this 80 kDa band is authentic DMPK. Many of the mAbs also recognize other proteins of higher or lower Mr, notably a 55 kDa protein, proteins of 200 kDa and a protein that migrates slightly faster than DMPK at 72 kDa [cross-reacting protein (CRP)]. Since these proteins are recognized by only one or two of 16 mAbs, they must be cross-reacting proteins with some sequence or structural similarity to DMPK. The argument against the lower Mr bands being alternatively spliced isoforms is that no mRNA splicing that removes the coil domain has been observed, so all nine mAbs against the coil domain should recognize all known splicing isoforms (13).
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Figure 1b and c shows that the 80 kDa DMPK band is not detectable in brain or lung, since many mAbs detect nothing at all. Some cross-reacting proteins are very prominent, especially in brain. mAb MANDM1 is the only one to detect a band close to the Mr of DMPK in lung and brain, but Figure 2a shows that this is a CRP of slightly lower Mr. The upper band of authentic DMPK is detected only in cardiac and skeletal muscle whereas CRP is also present in lung, brain and three different human skin fibroblast cell lines (F1–F3). Figure 2b shows that the upper band is also detectable by MANDM1 at very low levels in fetal stomach (smooth muscle), but this could not be detected by the whole panel of mAbs (data not shown), either because the other mAbs are weaker or because the band is not authentic DMPK. DMPK was also detected by the same panel of mAbs in fetal skeletal muscle, but not in fetal brain cortex, lung or liver (data not shown). Overall, the data suggest that DMPK is expressed mainly in skeletal and cardiac muscle. Lower levels may be present in smooth muscle, but DMPK levels were below the limits of detection in any other cell or tissue tested. DMPK mRNA has been detected by RT–PCR in non-muscle cells (7,29) so the possibility that DMPK protein is also produced at very low levels in non-muscle cells cannot be ruled out.
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In order to identify some of the cross-reacting proteins, a human brain cDNA library in the
Zap expression vector was screened with a mixture of mAbs which displayed strong non-DMPK bands on western blots of brain (Fig. 1b). Apart from several clones that reacted with all anti-DMPK mAbs (and presumably contained DMPK sequences), two clones were obtained that reacted with MANDM1 only. The first of these contained the human MRCKß sequence in the correct reading frame and identical to the published sequence (28; GenBank accession no. NM_006035). MRCKß has an Mr of 190 kDa, very close to the 200 kDa band recognized by MANDM1 in brain and other tissues (Fig. 1). The second clone contained MRCK catalytic domain sequence (Fig. 3a), identified by comparison with the published rat sequence (26). Recombinant full-length rat MRCK was used on a western blot to confirm binding of MANDM1 only and no other DMPK mAbs (data not shown). Figure 3 shows that MRCK is highly conserved between human and rat at both the mRNA and protein levels.
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Since the sequence of the protein kinase PK428 is identical to that of MRCK
, it may be a splicing isoform containing the complete catalytic domain (27). MANDM1 is therefore likely to recognize PK428 also. PK428 is reported to migrate on SDS–PAGE with an Mr of 65 kDa, so CRP in Figures 1 and 2 may be this protein kinase. It is possible, however, that PK428 is not detectably expressed as protein in any of the tissues tested here and that CRP is some other cross-reacting protein.
Detailed epitope mapping was attempted by using phage-displayed random peptide libraries. A library of bacteriophage fd-tet expressing 15mer peptides was selected with mixtures of anti-DMPK mAbs as described previously (30) and selected clones were tested for reaction with individual mAbs in the mixture. The expressed peptide sequence was identified by DNA sequencing and matched with the known DMPK sequence (Fig. 4). MANDM2 and MANDM4 both recognized the sequence FDLVxDG near the end of the catalytic domain [only 5 amino acids away from the alternatively spliced VSGGG sequence (13)], whereas the two 55 kDa mAbs, MANDM7 and MANDM8, recognized the nearby sequence (T)PD(F)E(G). Both sequences are significantly different in MRCK and -ß, explaining why these four mAbs do not cross-react with those proteins. GenBank was searched for TPDFEG (using Advanced Blastp with ‘word length = 2’ and ‘expectation = 10 000’), in the hope of identifying the 55 kDa protein, and two human proteins were found which contained PDFEG. One was sarcoplasmic reticulum calcium ATPase and the second was the P100 subunit of nuclear factor NF-
B (Fig. 4), but neither of these are 55 kDa proteins. MANDM1 appears to recognize the sequence RxIKxxxI in the middle of the catalytic domain and the RxIK sequence is conserved in MRCK and -ß, although not in Rho kinase, consistent with the observed cross-reactions of this mAb. The MANDM2/4 epitope sequence is altered in mouse DMPK (FDVVxDR instead of FDLVxDG; SwissProt accession no. AAC60666) whereas the MANDM1 epitope sequence is identical in mouse, consistent with the species specificities of these mAbs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our studies on the distribution of DMPK in human tissues have shown that DMPK is produced in substantial amounts only in heart and skeletal muscle. This conclusion agrees with in situ hybridization studies that showed DMPK mRNA in heart, skeletal muscle and, at lower levels, smooth muscle (31). Several early northern blot studies also showed that DMPK mRNA was present in both heart and skeletal muscle, but difficult to detect in other tissues (brain, lung, liver, etc.) (2,13,32). This suggests that DMPK defects may be involved in the cardiac and skeletal muscle problems in DM patients. This view is supported by the mild myopathy (31,33) and cardiac conduction defects (34) observed in DMPK knockout mice.
Several studies of DMPK protein using antibodies or of DMPK mRNA by RT–PCR, however, have suggested that DMPK is a widely distributed protein, found in many tissues and cultured cell lines. In an earlier study (25) we interpreted a 72–80 kDa doublet as DMPK, though it is now clear that only the upper 80 kDa band is authentic DMPK. Studies on rabbit tissues could only be done with MANDM1, since other mAbs are human specific and this led to confusion with CRP, which is abundant in non-muscle tissues (Fig. 2). This reinforces our assertion that only a panel of mAbs against different DMPK epitopes can ensure that authentic DMPK is being studied. The present work has been done with human tissues only, enabling us to use mAbs against at least four different catalytic domain epitopes and at least four different coil domain epitopes (based on epitope mapping and western blot cross-reactions). RT–PCR detection of DMPK mRNA in non-muscle tissues may be explained by the greater sensitivity of this technique compared with western blotting. However, prolonged exposure of the X-ray film for western blots still did not detect 80 kDa DMPK in non-muscle tissues (data not shown). An alternative possibility is that DMPK protein is more stable in muscle tissues and accumulates to higher levels. The presence of even small amounts of DMPK in non-muscle tissues would suggest that it has some function there, but the much higher levels in heart and muscle suggest a special function in these tissues.
Cross-reaction of mAb MANDM1 with MRCK and -ß has been confirmed unequivocally by its selection of these cDNAs from a human brain cDNA expression library and its binding to recombinant MRCK. The molecular basis for this cross-reaction is explained by MANDM1 selection of peptides containing RxIK from a phage-displayed random peptide library, a sequence which is shared by MRCK and -ß. This sequence is also shared by PK428, which is a likely splicing isoform of MRCK and a candidate for the lower Mr CRP band on western blots (Fig. 2), but is not shared by other related kinases, such as p160 Rho kinase (35–37). Although the three-dimensional structure of DMPK has not been determined, comparison with human cAMP-dependent protein kinase A (Brookhaven pdb accession no. 1YDT) shows that the MANDM1 epitope includes part of the conserved ‘catalytic loop’ which accepts protons from phosphorylatable Ser/Thr residues (HRDLKPEN in PKA and HRDIKPDN in DMPK) (38). Two mAbs which recognize only DMPK in human skeletal muscle, MANDM2 and MANDM4, selected a peptide containing FDLVxDG from the phage library and this sequence is not shared by the MRCKs. MANDM2 and MANDM4 differ in their exact epitope specificity, since MANDM2 cross-reacts with several proteins in human brain (Fig. 1b); this could be explained if MANDM4 tolerated less deviation from the epitope sequence than MANDM2. Identification of the MANDM7/8 epitope is of particular interest since these mAbs recognize a 55 kDa skeletal muscle-specific protein and react only weakly, if at all, with authentic DMPK (Fig. 1a) (25). Both mAbs recognize recombinant DMPK produced in bacteria, however, which suggests that post-translational modification may prevent recognition of DMPK from human muscle. The MANDM7/8 epitope lies within 20 amino acids of the alternatively spliced VSGGG sequence which is thought to influence post-translational modification (13). This linear epitope sequence (TPDFEG) is absent from other kinases, but PDFEG is highly conserved in sarcoplasmic reticulum calcium ATPases and is also found in human NF-B, suggesting two potential sources of cross-reaction. However, both of these proteins are larger than 55 kDa, so the identity of the 55 kDa protein remains unknown, although we have shown that it is adult specific, as well as skeletal muscle specific, being absent from 16 day fetal muscle (data not shown). Some workers have tried to avoid cross-reaction by using the coil and/or C-terminal domains as immunogens (19–24), since these regions have little sequence homology with known proteins. However, various protein bands were also detected by such antibodies and many mAbs against the coil domain in Figure 1 also show cross-reacting bands. We suggest that DMPK migrates on SDS–PAGE at 80 kDa, since it migrates between 97 and 60 kDa Mr markers and rather closer to the former. The increase over the predicted size of 69 kDa may be due to post-translational modification, possibly involving a glycosaminoglycan moiety and the unique VSGGG sequence in DMPK (13). Mr should not be used as a criterion for identification of authentic DMPK since its migration in relation to Mr markers may be affected by modification and electrophoresis conditions. In particular, our results do not exclude all ‘DMPK’ proteins previously reported as 70–74 kDa nor do they mean that all ‘DMPK’ protein bands described as 80–85 kDa are authentic. The demonstration of several different DMPK epitopes should be the only acceptable criterion.
It would clearly be valuable to use the mAbs that are monospecific on western blots (e.g. MANDM5) to localize DMPK in heart and skeletal muscle sections by immunofluorescence microscopy. We showed earlier that most of the mAbs stain intercalated discs in the heart, though rather faintly, but none of them show any specific localization in skeletal muscle (25). This suggests that the mAbs only recognize denatured DMPK on blots, but not native DMPK on frozen tissue sections. For example, in native DMPK the ‘catalytic loop’ sequence recognized by MANDM1 may be either inaccessible or unable to adopt the flexible structure which MANDM1 seems to recognize in free peptides and denatured DMPK. We have observed with another globular enzyme, creatine kinase, that mAbs tend to recognize either the native or the denatured form, but rarely both (39). Attempts to reveal DMPK by fixation or SDS treatment of muscle sections (40), however, have not yet been successful and new ‘native DMPK-specific’ mAbs may be required for immunolocalization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Production of mAbs
A panel of 12 mAbs against the catalytic and coil domains of DMPK has been described previously (25). A DMPK coil/C-terminal cDNA fragment, produced as previously described (25), was expressed as a thioredoxin fusion from pET32a and purified by His-tag affinity chromatography according to the supplier’s instructions (Novagen, Madison, WI). For immunization, it was precipitated with 50% ethanol and redissolved in 6 M urea in phosphate-buffered saline. A further four mAbs (MANDM13–16) were produced by immunization of BALB/c mice and fusion of spleen cells with Sp2/0 myeloma cells as described elsewhere (41). Hybridoma culture supernatants were tested by ELISA (41) and cell lines were established by two rounds of cloning by limiting dilution. All four mAbs were mapped to the coil domain using DMPK subfragments as previously described (25).
SDS–PAGE and western blotting
SDS–PAGE and western blotting were carried out essentially as described elsewhere (42). Antibody-reacting bands were visualized following development with a biotin–avidin detection system for mouse immunoglobulin (Vectastain kit; Vector Laboratories, Burlingame, CA) followed by a chemiluminescent detection system (SuperSignal; Pierce, Rockford, IL). Recombinant rat MRCK was a gift from T. Leung and L. Lim (University of Singapore, Singapore).
Screening cDNA libraries
A human brain cDNA library in the ZAP II vector (Stratagene, La Jolla, CA) was plated to produce plaques in a lawn of Escherichia coli, as described in the supplier’s protocols. Lifts on nitrocellulose were screened with mAb MANDM1 as described above for western blotting, except that diaminobenzidine was used as substrate. Positive plaques were amplified and cloned to homogeneity before recovery of the cDNA inserts in pBluescript plasmids, which were then subjected to dye-based automated DNA sequencing using plasmid primers (Cambridge Bioscience, Cambridge, UK). Novel human MRCK sequence was submitted to GenBank (accession no. AF250871).
Phage-displayed peptide library
A library of random 15mer peptides in the fuse5 filamentous phage was generously provided by George P. Smith (University of Missouri, Columbia, MO) and was subjected to reiterative selection with mAb mixtures as previously described (30). After cloning to homogeneity, peptide insert sequences recognized by individual mAbs were determined by dideoxy DNA sequencing using Sequenase (Amersham International, Little Chalfont, UK).
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ACKNOWLEDGEMENTS |
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We thank George P. Smith (University of Missouri) for the phage-display library, Thomas Leung and Louis Lim (University of Singapore) for recombinant MRCK
and the Muscular Dystrophy Campaign for research grants.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1978 293214; Fax: +44 1978 290008; Email: morrisge@newi.ac.uk
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REFERENCES |
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