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Human Molecular Genetics Pages 1957-1965  

Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies
Introduction
Results
Discussion
Materials And Methods
   Cloning and expression of DMPK cDNA fragments
   Production of monoclonal antibodies
   SDS-PAGE and western blotting
   Subcellular fractionation
   Immunofluorescence microscopy
Acknowledgements
References

Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies

Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies

Y. C. N. Pham, Nguyen thi Man, Le Thanh Lam and G. E. Morris*

MRIC Biochemistry Group, NE Wales Institute, Mold Road, Wrexham LL11 2AW, UK

Received July 2, 1998; Revised and Accepted August 18, 1998

There is considerable confusion in the literature about the size of the myotonic dystrophy protein kinase (DMPK) and its localization within tissues. We have used a new panel of monoclonal antibodies (mAbs) to begin to resolve these issues, which are important for understanding the possible role of DMPK in myotonic dystrophy. Antisera raised against the catalytic and coil domains of DMPK recognized a major 55 kDa protein and a minor 72-80 kDa doublet on western blots of human skeletal muscle. Ten mAbs, five against the catalytic domain and five against the coil region, recognized only the 72-80 kDa doublet. The 72 kDa protein was present in all tissues tested, whereas the 80 kDa component was variably expressed, mainly in skeletal and cardiac muscles. The 72 kDa protein was absent in a DMPK knockout mouse and was greatly increased in a transgenic mouse overexpressing human DMPK, confirming its identity as authentic DMPK. Two mAbs against the catalytic domain recognized only the more abundant 55 kDa protein, which was found only in skeletal muscle. Nine out of 10 mAbs located DMPK to intercalated discs in human heart, an affected tissue in myotonic dystrophy. However, co-localization of DMPK with acetylcholine receptors at neuromuscular junctions was not observed with any of the mAbs.Subcellular fractionation and sedimentation analysis suggest that a major proportion of the DMPK in skeletal muscle and brain is cytosolic.

INTRODUCTION

Myotonic dystrophy (DM) is an autosomal dominant disease caused by an unstable CTG repeat sequence in the 3[prime]-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene on chromosome 19 (1-3). The CTG repeat is present 5-35 times in the normal population and increases to between 50 and several thousand in DM patients. The age of onset and the severity are correlated with the size of the expansion, which tends to get larger in later generations within families, thus explaining the phenomenon of `anticipation' in DM (4). Nuclear retention of DMPK transcripts with expanded CUG repeats has been demonstrated (5) and large decreases in levels of DMPK poly(A)+ mRNA have been reported in DM fibroblasts and muscle biopsies (6,7). Abnormal localization of transcripts and disruption of nuclear function may account for the dominant `gain-of-function' effect of the triplet repeat expansion (7), just as abnormal localization in the nucleus of huntingtin protein with an expanded polyglutamine repeat may account for dominance in Huntington's disease (8).

The expanded CTG repeat at the DM locus is also within the 5[prime]-end of a nearby gene, DM-related homeobox protein (DMAHP), which is predicted to be encoded by a homeobox gene related to the `six' family (9). Effects on DMAHP expression 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 the heart (conduction defects) (10). A third gene, gene 59 (or DMR-N9 in the mouse), which lies 5[prime] of DMPK, might also be affected by very large CTG expansions which occur in severe cases (11).

The human DMPK cDNA has 15 exons and predicts a protein of ~70 kDa, though there may be variation in the size and sequence of the first exon (1-3,12,13). DMPK is a serine/threonine protein kinase in which the protein kinase catalytic domain (~43 kDa) is followed by a coiled coil domain (~12 kDa) and a hydrophobic domain which are not seen in any other protein kinases. Alternative splicing has been observed in the protein kinase domain (mouse only), in a glycosaminoglycan site between domains and in the C-terminal domain (14).

The history of attempts to characterize DMPK protein using antibodies is one of considerable confusion. Nearly all publications have reported quite large size variations between tissues for DMPK. Some of the problems have probably arisen because DMPK shares catalytic domain sequences with other protein kinases, with consequent cross-reaction of antisera. Anti-peptide antisera against the catalytic domain have recognized proteins of 52-55 kDa in skeletal muscle (15,16), 42/60 kDa in brain and heart (15,16) or 53 kDa in heart and skeletal muscle (17). The only monoclonal antibody yet produced (using recombinant catalytic and coil domains as immunogen) gave strikingly different results, recognizing a 64 kDa protein in muscle and a 79 kDa protein in brain (18). Using western blots of fibroblast extracts, however, several antisera prepared by Timchenko et al. (19) recognized a protein of the expected size (72 kDa), although one antiserum against a C-terminal domain peptide also recognized a 51-54 kDa protein in skeletal muscle (20). Variable results have also been obtained with antibodies against the C-terminal domain, which does not share sequences with any known protein. Polyclonal antisera raised against the whole C-terminal domain recognized proteins of 71-74 and 80-85 kDa in skeletal and cardiac muscle (21-23). However, a similar study produced antiserum recognizing proteins of 85 and 54 kDa (24) and an antiserum against a C-terminal peptide recognized proteins of 70 and 55 kDa in skeletal muscle (25). Finally, a 45 kDa protein was detected in brain using C-terminal antibodies (23).

Immunolocalization studies have shown DMPK antibodies binding to neuromuscular junctions in skeletal muscle (17) and to intercalated discs in the heart (21), but other studies suggest an association of DMPK with sarcoplasmic reticulum (24). It must be remembered that an antibody which recognizes a major band on western blots does not necessarily recognize the same protein in tissue sections, since some epitopes are exposed only by SDS treatment (26). As long as there is any suspicion of antibody cross-reaction, it is difficult to be confident about which bands on western blots are products of the DMPK gene and which localizations in tissue sections are authentic DMPK. Our approach to this problem has been 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. We argue that any protein on western blots which contains most, if not all, epitopes is likely to be authentic DMPK, whereas proteins sharing only one epitope with DMPK are likely cross-reactions. Similarly, an authentic localization of DMPK in tissue sections should be displayed by most, if not all, mAbs. We now describe the first results obtained with an mAb panel of this kind.

RESULTS

Recombinant immunogens were prepared by PCR amplification of two DMPK cDNA fragments from human skeletal muscle cDNA and cloning into bacterial expression plasmids. A fragment encoding the catalytic and coil domains (exons 2-12) was cloned into pMW172 and a second encoding the coil and C-terminal domains (exons 9-14) was cloned into pET32a, which produces a thioredoxin fusion protein. Nine mice were immunized with catalytic/coil domains and they all produced antisera which recognized a major band at 55 kDa and a minor doublet at 72 kDa in human skeletal muscle (Fig. 1). Three mice immunized with coil/C-terminal domains produced antisera recognizing the 72 kDa doublet only (data not shown). This observation alone suggests that the 72 kDa doublet is authentic DMPK and the 55 kDa protein is a cross-reactive protein sharing some sequence similarity with the DMPK catalytic domain.


Figure 1. Western blot of normal human muscle extract developed with antisera from nine mice immunized with partially purified recombinant DMPK protein. The extract was subjected to 3-12.5% gradient gel SDS-PAGE as a horizontal strip and the western blot was cut into vertical strips for analysis. Mr contains SeeBlue protein size markers (R&D Systems, Oxford, UK). Lanes 1-9 show antisera from nine mice used at 1:100 dilution. The blot was developed with the Vectastain second antibody system and DAB substrate. mAbs were produced from mice 4 and 8.

A panel of 12 mAbs (Table 1) against the catalytic/coil domains was produced from two separate hybridoma fusions and 10 out of 12 mAbs recognized only the 72 kDa protein doublet while two mAbs recognized only the 55 kDa protein (Fig. 2a). Epitope mapping showed that all mAbs recognize the catalytic/coil fusion protein on western blots (Fig. 3). Seven mAbs recognized the catalytic domain only (Fig. 3) while the other five mAbs recognized a fragment containing only the coil and C-terminal domains (data not shown). This confirms that the 72 kDa doublet is authentic DMPK since it contains mAb epitopes of both the catalytic and the coil regions. Both mAbs against the 55 kDa protein recognize the catalytic domain, supporting the possibility of a cross-reactive protein kinase. Using a sensitive chemiluminescent method for mAb detection on blots, we were not able to detect any cross-reaction of MANDM1 with a 55 kDa protein nor any cross-reaction of the 55 kDa mAbs with DMPK at 72 kDa (Fig. 2b).

As final confirmation that the 10 72 kDa mAbs recognize authentic DMPK, we have shown that the 72 kDa band in cardiac muscle is absent from a DMPK knockout (KO) mouse (27) and is greatly increased in a transgenic mouse overexpressing human DMPK (OE) (27; Fig. 4). Note that the mouse DMPK migrates as a single band, rather than a doublet. The lower 50 kDa band in the wild-type (WT) and KO lanes in Figure 4 may be a strain-specific cross-reaction, since it is not seen in the OE of a different strain. The much stronger staining of DMPK in OE compared with WT is not entirely due to overexpression of DMPK because MANDM1 binds human DMPK more strongly than mouse DMPK (cf. lanes NHM and WT in Fig. 4). No 55 kDa protein was detected by MANDM7 and 8 in mouse skeletal muscle overexpressing human DMPK (data not shown; note that the two 55 kDa mAbs do not cross-react with endogenous mouse 55 kDa protein).

Figure 5a shows that DMPK is found in a wide variety of rabbit tissues. It appears either as a single band at 72 kDa or, in cardiac muscle, as a doublet with an extra band at ~80 kDa. In rabbit brain, additional bands of both higher and lower Mr are seen. These were also seen in human brain with MANDM1 but not with the other mAbs (data not shown) and are therefore likely cross-reactions of MANDM1 only. The 80 kDa band is the major protein in rabbit heart, but we are unable to verify that this is all authentic DMPK because only MANDM1 recognizes rabbit DMPK. In human heart, however, where the 72 kDa band is the major component, both 72 and 80 kDa components are recognized by all 10 mAbs (Fig. 5b).

Table 1. Characterization of 12 mAbs against DMPK (catalytic + coil domains)
mAb Clone Ig subclass Main band on blots (kDa) Species specificity Domain recognized
MANDM1 6G8 G1 72 hu, rb, mo Catalytic
MANDM2 10A7 G1 72 hu Catalytic
MANDM3 8B5 G1 72 hu Coil
MANDM4 9H9 G1 72 hu Catalytic
MANDM5 5G12 G1 72 hu Catalytic
MANDM6 7E4B7 G1 72 hu Catalytic
MANDM9 3D10 G1 72 hu Coil
MANDM10 2H12 G1 72 hu Coil
MANDM11 6B6 G1 72 hu Coil
MANDM12 7E4G2 G1 72 hu Coil
MANDM7 12G5 G1 55 hu, rb Catalytic
MANDM8 7E7 G2a 55 hu, rb Catalytic
Ig subclass was determined using a mouse Ig typing kit (Serotec). The main band recognized on western blots is identified from Figure 2. Species specificity: hu, human; rb, rabbit; mo, mouse. Domain recognition was determined from Figure 3.

Simple subcellular fractionation was performed using rabbit brain and muscle tissues. After low speed centrifugation of a brain homogenate, DMPK was found in the supernatant and also in the nuclear pellet (Fig. 6). The DMPK in the pellet, however, appears to be in unbroken cells since it was solubilized by a buffer containing Triton X-100 (Fig. 6) and also by further homogenization in buffer without Triton (data not shown). The mitochondrial, or `heavy membrane', pellet contained little or no DMPK, but some was found in the microsomal pellet. Similar results were obtained with rabbit skeletal muscle except that, in this case, nearly all the DMPK remained in the soluble cytoplasmic fraction after high speed centrifugation (Fig. 6). The results suggest that most of the DMPK is soluble in low salt buffers and may be cytosolic. Figure 7 shows sucrose density gradient analysis of the post-mitochondrial supernatant from rabbit brain; this fraction contains nearly all the extractable DMPK. Most of the 72 kDa DMPK is found in four or five fractions at the top of the gradient. SDS treatment of the extract before loading onto the gradient shows that these fractions correspond to monomeric DMPK. A smaller proportion sediments rather broadly through the gradient and this is not altered by pre-treatment of the extract with Triton X-100 to solubilize membranes. The 72 kDa band in Figure 7 co-migrates on SDS-PAGE exactly with the 72 kDa DMPK band in human lung extracts (data not shown). MANDM1, the only mAb which recognizes rabbit DMPK, shows cross-reactions with three other proteins in rabbit brain. One of these remains at the top of the gradient, while the other two (one slightly smaller than DMPK at ~68 kDa) migrate in the gradient as large complexes of different sizes (Fig. 7). These internal controls show that the gradient system will detect large discrete complexes when they are present.


Figure 2. Western blot of normal human muscle extract developed with 12 DMPK mAbs. (a) The extract was subjected to 3-12.5% SDS-PAGE as a horizontal strip. Antibody analysis of the western blot was performed on a miniblotter apparatus (Immunetics, Cambridge, MA). mAbs loaded on the blot correspond to those in Table 1. The blot was developed with the Vectastain second antibody system and DAB substrate. (b) The blot was repeated for MANDM1 and 7 using a more sensitive chemiluminescent substrate instead of DAB to confirm that MANDM1 does not recognize the 55 kDa protein and MANDM7 does not recognize the 72/80 kDa doublet. On this particular gel the 55 kDa protein was resolved into two bands.


Figure 3. Domain mapping of 12 mAbs. Western blots of partially purified recombinant DMPK proteins containing (a) the catalytic and coiled coil domains (64 kDa) used for immunization and (b) the catalytic domain only (52 kDa). The fusion proteins were subjected to SDS-PAGE on 3-12.5% gradient gels as a strip and the blot was developed with 12 DMPK monoclonal antibodies (1:100 dilution) using a miniblotter apparatus and the Vectastain second antibody system with DAB substrate. Lane Mr contains Sigma prestained protein markers. The results were confirmed using a recombinant fusion protein containing the coil and C-terminal domains only (not shown).


Figure 4. The 72 kDa protein is abundant in a transgenic mouse overexpressing human DMPK and absent from a DMPK knockout mouse. A western blot of extracts of normal human muscle (control, NHM) and of hearts from the overexpressor (OE), wild-type (control, WT) and DMPK knockout (KO) mice was developed with mAb MANDM1 and peroxidase-conjugated rabbit anti-(mouse Ig) with Supersignal chemiluminescent substrate. Similar amounts of total protein were loaded for each of the three mouse heart extracts, as determined with a separate Coomassie blue stained gel (not shown). A 50 kDa cross-reacting protein is observed only in WT and KO mice (C57BL/129 strain) and not in the OE mouse (FVB strain).


Figure 5. Expression of DMPK in different rabbit tissues and human heart. (a) Extracts of rabbit skeletal muscle (Mu), heart (Ht), brain (cortex, BrCo; cerebellum, BrCe), spleen (Sp), liver (Li), kidney (Ki), spinal cord (SC) and sciatic nerve (SN) were subjected to SDS-PAGE on a 3-12.5% gradient gel and the western blot was developed with MANDM1, Vectastain second antibody system and DAB as substrate. (b) An extract of human heart muscle was subjected to SDS-PAGE as a horizontal strip and the western blot was analysed on a miniblotter with 12 mAbs. The major bands below 72 kDa are degradation products of DMPK.


Figure 6. Simple subcellular fractionation of rabbit brain and muscle. Fractions obtained by centrifugation were boiled with an equal volume of 2× SDS sample buffer and subjected to SDS-PAGE and western blotting with MANDM1 mAb (37). SN, supernatant; P, pellet. After centrifugation of the homogenate at 4000 g, the pellet (P4K) was resuspended in the original volume of buffer with 1% Triton X-100, sampled and then centifuged at 100 000 g to yield P4K post-Triton P and SN. The supernatant (SN4K) was further centifuged at 10 000 g to yield P10K and the 10 000 g supernatant (SN10K) was further centrifuged at 100 000 g to yield P100K and SN100K. In each case, pellets were resuspended in the same original volume to maintain comparability and an equal volume (5 µl) of each fraction was loaded for SDS-PAGE.

Figure 8 shows that MANDM1 stains intercalated discs in rabbit heart sections, in addition to a more general sarcoplasmic staining. In human heart sections, intercalated disc staining was observed with all 12 mAbs except MANDM2 (Fig. 9); it was not observed with control mAbs nor when the primary mAb was omitted (data not shown). Some mAbs recognize only denatured antigen on blots and not native antigen on sections; this may explain the result with MANDM2. Otherwise, the fact that mAbs against at least three different DMPK epitopes recognize intercalated discs is strong evidence for this localization. Intercalated disc staining was also observed with the two 55 kDa mAbs (MANDM7 and 8), even though the 55 kDa band is not present in heart. This suggests that these two mAbs recognize native DMPK on tissue sections (and recombinant DMPK in ELISA) but not denatured DMPK on western blots after SDS-PAGE. None of the 12 mAbs stained neuromuscular junctions (NMJs) in sections of human skeletal muscle (three examples, including one of the 55 kDa mAbs, are shown in Fig. 10), although a control mAb against utrophin, an established NMJ protein, does show the required co-localization with acetylcholine receptors (Fig. 10). We are therefore unable to confirm previous reports of NMJ localization with these mAbs. Figure 11 shows that some weak staining of rabbit intercostal muscle in the region of NMJs was observed with MANDM1 but this does not co-localize with acetylcholine receptors in the same way as dystrophin (not shown) or utrophin (Fig. 10) controls. mAb staining of intercalated discs was also observed with acetone/methanol fixed sections, but NMJ staining was still not observed after this fixation method (data not shown). Formalin fixation appears to destroy the DMPK epitopes since intercalated disc staining was lost after formalin treatment (data not shown).


Figure 7. Sucrose density gradient analysis of 10 000 g supernatants from rabbit brain. An aliquot of 1 ml of a 10 000 g supernatant from a rabbit brain homogenate (obtained as in Fig. 6) was loaded onto a 26 ml linear gradient of 5-20% sucrose in RSB buffer, either (a) untreated, (b) after addition of Triton X-100 to 1% final concentration or (c) boiling for 2 min with 1% SDS (final concentration). Every other fraction (i.e. 1, 3, 5, etc.) was analysed by SDS-PAGE and western blotting with MANDM1 mAb.

DISCUSSION

We have shown that 10 mAbs against at least three different epitopes in the coil and catalytic domains of DMPK recognize a protein doublet of ~72 and 80 kDa in skeletal and cardiac muscle. This is strong evidence that this protein is authentic DMPK and is supported by the 70 kDa Mr predicted from the cDNA sequence. In non-muscle tissues and cell lines, the 72 kDa form clearly predominates. Other protein bands were detected by MANDM1, notably in brain, but these were not detected by all 10 mAbs and are probably cross-reacting proteins. Northern blot data have shown that DMPK mRNA is present in all tissues, but is more abundant in muscle (14). We have also found DMPK protein in all tissues examined (Fig. 6), though its abundance in brain seems as high as in skeletal and cardiac muscle tissues, with only slightly lower levels in liver and kidney. However, protein levels depend on turnover rates as well as on mRNA levels. The presence of a doublet with a higher Mr band of ~80 kDa in some, but not all, tissues could be explained by alternatively spliced isoforms (12,14), post-translational modification (e.g. phosphorylation) or conformational differences (23). Two other groups have described a double band in skeletal muscle using antisera against the C-terminal domain, e.g. 71 and 80 kDa in Maeda et al. (21) or 74 and 82 kDa in Whiting et al. (23), and it seems likely that these correspond to the protein doublet reported here. Whiting et al. (23) also observed predominance of the lower Mr component of the doublet in human heart and the higher Mr component in rat heart, exactly as we found when comparing human and rabbit hearts in Figure 5. Since these two proteins are recognized by antibodies against the catalytic and coil domains (this study) as well as the C-terminal domain (21,23), they are likely to have most, if not all, of the DMPK sequence.


Figure 8. DMPK at intercalated discs in rabbit heart. (A) mAb MANDM1. (B) Phase contrast microscopy. Three of the many intercalated discs present are indicated by arrows.


Figure 9. DMPK is located at intercalated discs in human heart by 11 of 12 mAbs. For each mAb (MANDM1-12), one of the intercalated discs is indicated by an arrow (except MANDM2 which did not stain discs).

Two groups using the same C-terminal anti-DMPK antiserum have localized DMPK to intercalated discs in heart and to NMJs in skeletal muscle (21,23). We have confirmed the intercalated disc localization, both with mAbs against the catalytic domain and with mAbs against the coil domain. This strong and consistent intercalated disc staining is highly specific and characteristic of DMPK, since we have not observed such staining with mAbs against dystrophin or utrophin (data not shown). We could, however, find no evidence for close association of DMPK with NMJs. This is surprising in view of the indirect supportive evidence for NMJ localization provided by the recent studies of Suzuki et al. (28) which found that a heat shock protein which binds DMPK is partly localized at NMJs. Weak localization in the NMJ region as shown in Figure 11 is the nearest we could get, after making some effort in view of the previous published evidence, but even here it is quite clear that there is no precise co-localization with acetylcholine receptors. It is often argued that localization of DMPK at intercalated discs or NMJs may account for cardiac conduction defects and myotonia, respectively, in DM, although no loss of DMPK from either NMJs or intercalated discs has yet been demonstrated in DM patients. Furthermore, other genetic mutations causing myotonia have been found in ion channel proteins (29), rather than in NMJ proteins. Antibody studies cannot demonstrate a complete absence of DMPK from NMJs, but we may say with some certainty that the concentration of DMPK is much lower at NMJs than at intercalated discs. The possibility that DMPK is only weakly bound to NMJs and is washed out of unfixed tissue sections was also considered, but similar results were obtained with acetone/methanol fixed sections. Evidence for sarcoplasmic reticulum localization of DMPK from immunohistochemistry and biochemical fractionation has also been reported (18,24,25) and we have occasionally observed immunolabelling of cross-striations in cardiac muscle, but not consistently (Fig. 9). Our sucrose gradient analysis shows that the bulk of the extractable DMPK in rabbit brain remains at the top of the gradient, consistent with monomeric protein, dimers or small oligomers, but not with large complexes (Fig. 7). A small proportion which migrates further down the gradient would be consistent with larger heterogeneous complexes. The lack of effect of Triton X-100 treatment before centrifugation suggests that these faster sedimenting particles are not membrane fragments (microsomes). Once again, although the results suggest that the bulk of the DMPK in muscle (Fig. 6) and brain (Figs 6 and 7) is not membrane bound, the possibility of a small proportion being associated with membranes cannot be ruled out. It might be argued that polyclonal antibodies against DMPK stain NMJs or sarcoplasmic reticulum because they are stronger than our mAbs, but this explanation is not supported by the strong staining of intercalated discs by the mAbs (Figs 8 and 9).


Figure 10. DMPK is not detected at neuromuscular junctions in human skeletal muscle. Double-labelling was performed using FITC-[alpha]-bungarotoxin ([alpha]-BT) to label acetylcholine receptors. Localization of utrophin at NMJs is observed (MANCHO7 mAb, arrows), but not DMPK (three selected mAbs, MANDM8, 4 and 1, are shown).

There are many reports in the literature of anti-DMPK antisera which recognize 52-55 kDa proteins, especially when the immunogen, as in the present case, includes the catalytic domain. It is clear that the 55 kDa protein recognized by polyclonal antisera in Figure 1 might have been mistaken for DMPK, if mAbs had not been prepared to distinguish the two proteins. The 55 kDa protein may simply share an epitope with DMPK, either by chance or because of a functional relationship; for example, many protein kinases may share sequences with the DMPK catalytic domain (30-32). However, 52-55 kDa proteins have also been detected with antisera against C-terminal peptides (20,25) and 52-55 kDa proteins have been detected in heart and brain (12,15,17) and fibroblasts (19), whereas our 55 kDa protein is skeletal muscle specific. For this reason, our 55 kDa protein cannot account for all previous reports of a DMPK of this size. The possible existence of a 52-55 kDa protein kinase which is closely related to DMPK and has tissue-specific isoforms cannot be ruled out. Our 55 kDa protein does not appear to be one of the alternatively spliced isoforms of DMPK since antibodies against the coil domain do not recognize it and removal of the coil domain by splicing has not been reported (12-14). A degradation product of the 72 kDa DMPK is ruled out by the mAb specificity, tissue specificity and abundance of the 55 kDa protein. The two mAbs against the 55 kDa protein do recognize the recombinant DMPK antigen, so it is remarkable that they do not seem to recognize DMPK on western blots. There is a possibility that they recognize native DMPK in tissue sections (Fig. 9); consequently, these mAbs cannot be used reliably for localization of either the 55 kDa protein or DMPK itself. This unusual type of cross-reaction is not unprecedented; an mAb raised against dystrophin was found to recognize only [alpha]-actinin in muscle sections, though it recognized both dystrophin and [alpha]-actinin on western blots (33). In this case, the common epitope was later found to be folded into an [alpha]-helix in dystrophin only, probably accounting for its inaccessibility in native dystrophin in tissue sections (34). The cross-reaction of MANDM7 and 8 will only be fully understood when the epitope is characterized and the 55 kDa protein sequence is determined.

In conclusion, our studies with the first panel of mAbs against different DMPK epitopes clearly demonstrate an association of DMPK with cardiac intercalated discs, although most DMPK appears to be cytoplasmic in muscle and brain.


Figure 11. Faint staining at rabbit intercostal muscle membranes by MANDM1 does not co-localize exactly with acetylcholine receptors (AChR).

MATERIALS AND METHODS

Cloning and expression of DMPK cDNA fragments

PCR primers were modified from those of Dunne et al. (35) by incorporating restriction sites for cloning into pET vectors. The primers were: (i) for the catalytic/coil domains, forward 5[prime]-gcgggatccatcgtggtgaggctt and reverse 5[prime]-gacaggcctgacagctgtggctcc with BamHI and StuI sites respectively for cloning into pMW172 (36); (ii) for the catalytic domain only in pMW172, the same forward primer with 5[prime]-gcgaggcctcgtgtggatcaagcag as reverse primer; (iii) for the coil/C-terminal domains, forward 5[prime]-gcgaattcctgcttgagccacac and reverse 5[prime]-cgaagcttcagtcttccaacgg with EcoRI and HindIII sites respectively for cloning into pET32a (Novagen).

After PCR under standard conditions (37) using total human muscle cDNA (Clontech) as template, the PCR product was purified using QIAquick (Qiagen) and cloned into the pT7Blue T-tailed vector (Novagen). After verifying the sequence using Sequenase 2.0 (Amersham International), the DMPK cDNA was sub-cloned into pMW172 or pET32a followed by electroporation into Escherichia coli BL21(DE3) as described previously (37). Optimal recombinant protein expression was obtained by induction with 1 mM IPTG for 16 h at 37°C.

Catalytic/coil domain protein from pMW172 was partially purified from inclusion bodies by sequential extraction with increasing concentrations of urea in phosphate-buffered saline (PBS). Protein extracted with 4 M urea was used for immunization. The coil/C-terminal domain was expressed as a thioredoxin fusion from pET32a and was purified by His tag affinity chromatography according to the supplier's instructions (Novagen); for immunization, it was precipitated with 50% ethanol and redissolved in 6 M urea in PBS.

Production of monoclonal antibodies

mAbs were produced by immunization of BALB/c mice and fusion of spleen cells with Sp2/0 myeloma cells as described elsewhere (38). Hybridoma culture supernatants were tested by ELISA (38) and 12 cell lines were successfully established after two rounds of cloning by limiting dilution.

SDS-PAGE and western blotting

SDS-PAGE and western blotting were carried out essentially as described elsewhere (39). Antibody reacting bands were visualized following development with a biotin/avidin detection system for mouse immunoglobulin (Vectastain kit; Vector Laboratories) followed by diaminobenzidine (DAB) (Sigma) or with peroxidase-labelled rabbit anti-(mouse Ig) (DAKOpatts) followed by a chemiluminescent system (SuperSignal; Pierce).

Subcellular fractionation

Rabbit skeletal muscle and brain tissues (0.5 g) were homogenized in ice-cold RSB buffer (0.01 M NaCl, 0.0015 M MgCl2, 0.01 M Tris-HCl, pH 7.5) using a Dounce hand homogenizer for brain and a Silverson blender for muscle. All stages were carried out at 4°C. The homogenate was centrifuged at 4000 g for 10 min. The supernatant was centrifuged at 10 000 g for 10 min. The 10 000 g post-mitochondrial supernatant was further centrifuged at 100 000 g for 30 min. The 4000 g pellet of nuclei and unbroken cells was further extracted with 1% Triton X-100 in RSB and centrifuged at 100 000 g for 30 min.

Immunofluorescence microscopy

Unfixed, frozen sections of human and rabbit tissues (6 µm) were mounted on glass slides and stored at -70°C. FITC-labelled anti-(mouse IgG) (DAKOpatts) was used to detect bound antibody as described previously (39). For double label experiments, TRITC-labelled anti-(mouse Ig) was used with FITC-labelled [alpha]-bungarotoxin (Sigma) as described previously (39). Where stated, sections were fixed by exposure to methanol/acetone (50/50 v/v) for 2 min or by exposure to 4% formalin in PBS (pH 7.2) for 10 min, followed by 1 M glycine in PBS for 10 min.

ACKNOWLEDGEMENTS

We thank Tracy Fitzgerald for skilled technical assistance, Caroline Sewry (Imperial College School of Medicine, UK) for providing sections of human heart and of human skeletal muscle with NMJs, the HGMP Resource Centre (Cambridge, UK) for providing some of the PCR primers, John Kendrick-Jones (MRC Cambridge, UK) for pMW172 and Patricia Groenen and Be Wieringa (Nijmegen, The Netherlands) for tissue extracts from transgenic mice. This work was supported by grants from the Muscular Dystrophy Group of Great Britain and Northern Ireland and the British Heart Foundation.

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*To whom correspondence should be addressed. Tel: +44 1978 293330; Fax: +44 1978 290008; Email: morrisge@newi.ac.uk

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