© 2000 Oxford University Press Human Molecular Genetics, 2000, Vol. 9, No. 14 2167–2173 Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart L.T. Lam, Y.C.N. Pham, Nguyen thi Man and G.E. Morris+ 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 Myotonic dystrophy (DM) is a multisystemic disorder ness), brain (mental retardation), eyes (cataracts), testis caused by an inherited CTG repeat expansion which (atrophy) and heart (conduction defects) (10). It is important, affects three genes encoding the DM protein kinase therefore, to establish the normal tissue distribution of all three (DMPK), a homeobox protein Six5 and a protein proteins produced from the DM locus. containing WD repeats. Using a panel of 16 mono- The human DMPK cDNA has 15 exons and predicts a ∼ clonal antibodies against several different DMPK protein of 70 kDa, although there may be variation in the size epitopes we detected DMPK, as a single protein of and sequence of the first exon (1–3,11,12). DMPK is a serine/ threonine protein kinase in which the catalytic domain ∼80 kDa, only in skeletal muscle, cardiac muscle and, (∼43 kDa) is followed by a coiled-coil domain (∼12 kDa) and to a lesser extent, smooth muscle. Many earlier a hydrophobic C-terminal domain. Alternative splicing has reports of DMPK with different sizes and tissue distri- been observed in a short VSGGG sequence between domains butions appear to be due to antibody cross-reactions and in the C-terminal domain (13). Characterization of DMPK with more abundant proteins. One such antibody, protein using antibodies has proven to be very difficult and is MANDM1, was used to isolate two related protein still a source of considerable confusion. This may be largely kinases, MRCKα and β, from a human brain cDNA due to antibody cross-reaction, to which DMPK antibodies library and the shared epitope was located at the seem to be particularly vulnerable. Anti-peptide antisera against catalytic site of DMPK using a phage-displayed the catalytic domain have recognized proteins of 52–55 kDa in random peptide library. The peptide library also iden- skeletal muscle (14,15), 42 and 60 kDa in brain and heart tified an epitope shared between DMPK and a 55 kDa (14,15) or 53 kDa in heart and skeletal muscle (16). Several antisera prepared by Timchenko et al. (17) did recognize a muscle-specific protein. The results suggest that protein of the expected size (72 kDa) in fibroblasts. A mono- effects of the repeat expansion on the DMPK gene clonal antibody produced using recombinant catalytic and coil may be responsible for muscle and heart features of domains as immunogen recognized a 64 kDa protein in muscle DM, whereas clinical changes in other tissues may be and a 79 kDa protein in brain (18). However, variable results due to effects on the other two genes. 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 INTRODUCTION domain recognized proteins of 71–74 and 80–85 kDa in skel- Myotonic dystrophy (DM) is caused by an unstable CTG etal and cardiac muscle (19–21). However, a similar study repeat sequence in the 3′-untranslated region of a protein produced antiserum recognizing proteins of 85 and 54 kDa kinase (DMPK) gene on chromosome 19 (1–3). The CTG repeat (22) and antisera against C-terminal peptides recognized proteins increases from 5–30 in the normal population to 50–1000+ in of 70–72 and 50–55 kDa in skeletal muscle (23,24). Finally, a DM patients and the increase is correlated with disease 45 kDa protein was detected in brain using C-terminal antibodies severity (4). Nuclear retention of DMPK transcripts with (21). expanded CTG repeats has been demonstrated (5) and The correct approach to this problem is to prepare a panel of decreased DMPK poly(A)+ mRNA levels have been reported monoclonal antibodies (mAbs) which recognize several in DM tissues (6,7). The expanded CTG repeat at the DM locus different epitopes on DMPK. Although mAbs, like antisera, is also within the 5′ end of the Six5 gene predicted to encode a often display cross-reactions with other proteins, these cross- homeobox protein (8) and a third gene, DMWD (gene 59, or reactions tend to be very specific for each epitope. Any protein DMR-N9 in the mouse), which lies 5′ to DMPK,isalso on western blots which contains most, if not all, of the epitopes affected by CTG expansions (9). Effects on expression of non- is likely to be authentic DMPK, whereas proteins sharing only DMPK proteins could therefore be responsible for some, if not one epitope with DMPK are likely cross-reactions. Our initial all, of the clinical features of DM. DM affects many different results with a panel of 12 mAbs against the catalytic or coil tissues, including muscle (myotonia and progressive weak- domains suggested that DMPK was expressed as 80 and +To whom correspondence should be addressed. Tel: +44 1978 293214; Fax: +44 1978 290008; Email: [email protected] 2168 Human Molecular Genetics, 2000, Vol. 9, No. 14 Table 1. Sixteen mAbs against DMPK catalytic or coil domains mAb Clone no. Species specificitya Domain recognizedb MANDM1 6G8 hu, rb, mo Catalytic MANDM2 10A7 hu Catalytic MANDM3 8B5 hu Coil MANDM4 9H9 hu Catalytic MANDM5 5G12 hu Catalytic MANDM6 7E4B7 hu Catalytic MANDM7 (55 kDa) 12G5 hu, rb Catalytic MANDM8 (55 kDa) 7E7 hu, rb Catalytic MANDM9 3D10 hu Coil MANDM10 2H12 hu Coil MANDM11 6B6 hu Coil MANDM12 7E4G2 hu Coil MANDM13 12A4 hu Coil MANDM14 7E6 hu Coil MANDM15 5C9 hu Coil MANDM16 9A4 hu Coil ahu, human; rb, rabbit; mo, mouse. bDomain recognition was determined using recombinant DMPK fragments encoded by exons 2–8 (catalytic) or exons 9–12 (coil) (25). 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 Figure 1. Western blot detection of DMPK and cross-reacting proteins in 80 kDa protein is authentic DMPK and that it is detectable only (a) skeletal muscle, (b) lung and (c) cerebral cortex, using a panel of 15 mAbs. in skeletal muscle, in cardiac muscle and, at lower levels, in DMPK is found only in skeletal muscle (a). Human tissue extracts were pre- pared by homogenization and boiling in 1% SDS buffer before separation as a smooth muscle. Cross-reacting proteins of 190 and 72 kDa strip on 3–12.5% polyacrylamide gradient gels as described previously. The recognized by mAb MANDM1 appear to be isoforms of western blot on nitrocellulose was cut into vertical strips, each of which was myotonic dystrophy-related cdc42-binding kinase (MRCK) incubated with a different mAb (MANDM mAb numbers at top of blot; C, no- (26–28). mAb control; MANDM12 was omitted). The strips were reassembled in the presence of chemiluminescent substrate for exposure to X-ray film. Mr, bioti- nylated markers (catalase, 60 kDa; phosphorylase, 97 kDa); MRCK, myotonic RESULTS dystrophy-related cdc42-binding kinase; CRP, cross-reacting protein. A panel of 12 mAbs, prepared using a DMPK fragment containing the catalytic and coil domains, has been described alternatively spliced isoforms is that no mRNA splicing that previously (25). Seven mAbs recognized the catalytic domain removes the coil domain has been observed, so all nine mAbs and five recognized the coil domain. A further four mAbs, against the coil domain should recognize all known splicing MANDM13–16, were prepared using recombinant coil plus isoforms (13). C-terminal domains and all four of these mapped to the coil Figure 1b and c shows that the 80 kDa DMPK band is not domain (Table 1). detectable in brain or lung, since many mAbs detect nothing at Figure 1a shows that the only protein recognized by nearly all. Some cross-reacting proteins are very prominent, espe- all mAbs on a western blot of human skeletal muscle is a single cially in brain. mAb MANDM1 is the only one to detect a band ∼ band of 80 kDa. Since these mAbs recognize several different close to the Mr of DMPK in lung and brain, but Figure 2a epitopes and they all recognize the DMPK recombinant fusion shows that this is a CRP of slightly lower Mr. The upper band protein (data not shown), this 80 kDa band is authentic DMPK. of authentic DMPK is detected only in cardiac and skeletal Many of the mAbs also recognize other proteins of higher or muscle whereas CRP is also present in lung, brain and three ∼ lower Mr, notably a 55 kDa protein, proteins of 200 kDa and different human skin fibroblast cell lines (F1–F3). Figure 2b a protein that migrates slightly faster than DMPK at ∼72 kDa shows that the upper band is also detectable by MANDM1 at [cross-reacting protein (CRP)]. Since these proteins are recog- very low levels in fetal stomach (smooth muscle), but this nized by only one or two of 16 mAbs, they must be cross- could not be detected by the whole panel of mAbs (data not reacting proteins with some sequence or structural similarity shown), either because the other mAbs are weaker or because to DMPK. The argument against the lower Mr bands being the band is not authentic DMPK. DMPK was also detected by Human Molecular Genetics, 2000, Vol.