PRUEBAS DINÁMICAS ESTABLES

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Mupa-1 Mupa-2 Urd40 Mancho-3 Mupa-3 Mupa-2 Mupa-3

^C57BI UK9Ç57BI UKO,

Mupa-1 Mupa-2

Fig 4.4 Western blot analysis of kidney and lung extracts of C57BI and UKO®^mice (UKO). A, adult using Mupa-1, Mupa-2, Mupa-3, Urd40 and Mancho-3 antibodies. B, 16.5dpc embryos using Mupa-2 and Mupa- 3 antibodies. C, adult lung extracts immunobloted with Mupa-1 and Mupa-2.

§ category A, ^category B, *category D (Table 4.1)

utrophin : 4 B-spectrin: 49 Utrophin: 63 fi-spectrin: 108 Utrophin: 123 B-spectrin: 168 Utrophin: 183 B-spectrin: 228

SD EHMDVQKKTF TKWINÀRF SKS GKP PI SDHFXXXmXX>:X>mXXXXmPKE- RGS 62

+DE VQKKTFTKU+N+ ++ I+D++ PK +G

ADEREAVQKKTFTKWN^LARVSCR-ITDLYTDLPDGRMLIKLLEVLSGERLPKPTKGR 107 T RVHALMVUPy L Q VLHQHHVD L VHIGGTDIVÀGNPKL T L GL LWS11 LfflJQ VKD VHKDIH 12 2

R+H L W++ LQ L + V L N+G DI7 GN +LTLGL+U+IIL +Q++D+ +

+ + + + + + LL U + T Y VÏÏ+ NFTTSW DG+ÀFNÀ++H+H+PDL D+D++ K

L +ÀF+ A LG+ KLL PE ++V PD+KSII Y+ + +

Fig 4.5 aa alignment between mouse utrophin aminoacids

24 to 320 used to raise Mupa-1 and mouse R-spectrin, non-erythroid (accession: Q62261). The areas in blue indicate aa sequences where there is some similarity between the two proteins. += conservative substitution x= residue masked by BLAST to prevent artefactual hits

- =insertion or deletion of a single aa

p-spectrin and utrophin are both members of the dystrophin/spectrin

superfamily (Tinsley et. al, 1993). Mouse p-spectrin non-erythrocytic (the murine homologue of the human p-spectrin non-erythrocytic 1 ) is the best candidate for this polypeptide since its molecular weight is 274kDa. a-actinin is another member of the dystrophin/spectrin superfamily but was excluded from considerations since it has a mw of lOSkDa. p-spectrin is most abundant in brain and is also expressed in kidney and in all tissues in which a band of this size is detected by Mupa-1 including lung, muscle and heart.

While the likelihood of the 260kDa band being p-spectrin is high, the alternative explanation that it is a novel utrophin isoform with the same NH2-

terminus but terminating prior to the aa sequence corresponding to Urd40 and Mupa-2 might be considered. An argument against this view is that if this

hypothetical novel transcript contained sequence corresponding to the targeted exon in UKO®^® mice it would not be translated in UKO®^® samples as is the case for full-length utrophin. Furthermore, no evidence of such an unusual isoform was found by RT-PCR (chapter 5 section 5.3).

The 1 lOkDa polypeptide detected in adult and foetal kidney (Fig 4.4) by Mupa-2 only was placed in the same category as the 260kDa band (category B). However, in this case no good candidate proteins were identified in the protein databases which could be responsible for crossreactivity. Such a

candidate may emerge as data analyses from the Human Genome Sequencing project are completed.

Bands of 160kDa and 140kDa are detected with all the antibodies but occur only in control tissue. These bands are interpreted as degradation products of full-length utrophin (category A) since they are absent in UKO®^®

kidney and other UKO®^® tissues, but are detected in kidney and all control tissues rich in full-length utrophin. If this interpretation is correct then it suggests that degradation occurs in such a way that the breakdown products retain the actin binding domain intact and are preferentially detected with Mupa-1. A ladder of bands is also seen in association with the 260kDa component which suggests that p-spectrin is also susceptible to degradation. As might be predicted, in this case, breakdown products are also seen in UKO®^® samples where p-spectrin is expressed as in control tissues.

Another weak possibility is that the 160kDa and 140kDa bands are genuine short utrophin isoforms (Up140 predicted mw is 147 kDa) which are lost in the absence of full-length utrophin. This could be explained if for example they were involved in a membrane protein complex alongside full-length

utrophin and the DAPs; when the complex no longer assembles, as in UKO®^® mice, these isoforms are no longer stable and degrade. This hypothesis seems unlikely because the 140kDa/160kDa bands are detected, albeit weakly, by Mupa-1, the NH2-terminal antibody, (Fig 4.4 A and C). Genuine short isoforms,

would not contain the epitope corresponding to the actin-binding domain, whereas some breakdown products of full-length utrophin might contain such sequences. Thus, the best explanation is that these bands are degradation products of full-length utrophin but other scenarios cannot be excluded.

One band of 120 kDa was placed in category D (Fig 4.4 A and B) as a promising candidate for short utrophin isoform. This band was detected with all the antibodies except for the NH2-terminal antibody Mupa-1 and was

consistently present in both control and UKO®^® adult and foetal kidney. It was not detected in any other tissue and thus appears to be a short utrophin isoform

that is kidney specific. A band of similar mw was reported by Nguyen et al. (1995) in a kidney derived cell line (COS) extract and by Rafael et al. (1999) in the kidney of the double dystrophin/utrophin knock-out mouse (dko®^®) using

Mancho-3. In their illustration the 120 kDa band is missing from the control kidney. However, careful scrutiny of Figs shown in Loh et al. (2000) finds a component of c. 120kDa in control kidney extracts after immunoprécipitation with Urd40.

It is interesting to note that in the adult kidney (Fig 4.4 A), this 120kDa polypeptide appears upregulated in UKO®^® compared to the control sample and this is most evident with Urd40 and Mancho-3. 5'RACE was carried out in an attempt to isolate the transcript corresponding to this short isoform from adult kidney. These experiments are described later in this chapter in sections 4.4 and 4.5.

In summary, immunoblot analysis of mouse kidney extracts suggests that at least two different utrophin proteins might be co-expressed in this tissue, full- length utrophin and a 120kDa novel short utrophin isoform.

Brain Expression of full-length utrophin RNA and protein in the

mouse brain is well documented (Blake et al., 1995; Khurana et al., 1992; Kamakura et al., 1994; Lumeng et al., 1999). There is good evidence from mRNA in situ hybridisation experiments and Northern blotting for the presence of the short transcript, G-utrophin, in specific areas of the mouse and rat brain and in the trigeminal and dorsal root-ganglia (Blake et al., 1995; Knuesel et al., 2000). There is also some data from RT-PCR about the expression of D p i40 and Up71 in adult and foetal mouse and human brain (Wilson et al., 1999).

However, information about whether these transcripts are being translated in neuronal tissues is controversial. Fabbrizio et al. (1995) and Lumeng et al. (1999) both reported bands on immunoblots which could correspond to Up71. These occur in rabbit sciatic nerve and mouse brain extracts respectively and were detected using two different anti-human and anti-mouse COOH-terminal antibodies , K5B1 and COOH-ter respectively (see Fig 4.2 A). The authors did not detect G-utrophin in these tissues. In contrast, Knuesel et al. (2000)

detected high levels of a polypeptide, the correct size for G-utrophin, in rat brain using a polyclonal antibody against the distal rod domain of rat utrophin, Ut43, (see Fig 4.2 A) but failed to detect any bands that might correspond to Up71.

Using the Mupa antibodies on Western blots, full-length utrophin was detected in adult and foetal C57BI brain extracts but was absent from UKO®’'® samples. In Fig 4.6, full-length utrophin can be seen (395kDa) in control brain with Mupa-1, Mupa-2, Mupa-3 and Mancho-3 but is not seen in UKO®^® brain. Note that full-length utrophin appears as a very weak band with Mupa-1 in comparison to that seen with Mupa-2 and Mupa-3, a situation very similar to that described in kidney. Overall, brain expresses lower levels of full-length utrophin than kidney and lung but higher levels than muscle. Brain shows a similar complexity of bands as kidney and the bands were categorised as described previously (Table 4.1) in order to aid the interpretation of the banding patterns.

A 260 kDa polypeptide is detected only with Mupa-1 in control and UKO®^® extracts and here, as in kidney, it probably corresponds to mouse p- spectrin non-erythrocytic which is preferentially expressed in brain (category B).

39SkDa ^

t260kDa— ^

w

# #

IIO k D a *

*65kD a mamm 66 kDa +

C57BI UKO C57BI UKO C57BI UKO C57BI UKO