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Figure 1 | BMC Developmental Biology

Figure 1

From: Dominant negative Bmp5mutation reveals key role of BMPs in skeletal response to mechanical stimulation

Figure 1

Bmp5clv, a Bmp5 cleavage sequence mutation, disrupts the proteolytic processing of the BMP5 protein. (a) Schematic of the Bmp5 open reading frame showing the leader signal (LEAD), pro (PRO) and mature (MAT) domains and the putative proteolytic processing site (scissors). The G-to-A mutation (*) at nucleotide 932 of the Bmp5clvallele is predicted to destroy a Taq1 restriction site and to disrupt the first arginine residue in the putative conserved "RXXR" cleavage sequence in BMP5 (box). "1" and "2" denote positions of PCR primers used in typing the Bmp5clvallele. (b) Partial sequence traces showing the G-to-A substitution (*) in Bmp5clvmutant mice. This alteration is the only nucleotide difference in the Bmp5 coding region between wild-type (wt) and Bmp5clvmice. (c) Confirmation of mutation in genomic DNA. A 158-bp PCR product (gray arrowhead) containing the site of the Bmp5clvmutation is cleaved by Taq1 into 67- and 91-bp fragments (black arrowheads) in wild-type mice, partially cleaved in Bmp5clv/+ heterozygous mice, and not cleaved in Bmp5clv/Bmp5clvmice. (d) COS-7 cells were transfected with a mammalian expression vector (vec) or the same vector driving expression of wild-type (wt) or mutant (Bmp5clv) BMP5 protein. Secreted proteins were analyzed by Western blot with antibodies against the pro (anti-PRO) or mature (anti-MAT) domain of murine BMP5. Most of the wild-type protein expressed was in the smaller cleaved form, whereas all the detectable mutant protein was non-processed. No appreciable signal was detected by either antibody in the control cells. (e) A proposed dominant-negative mechanism for the Bmp5clvmutation. Wild-type BMP5 peptides are cleaved at the proteolytic site to form functional dimers with another wild-type copy of BMP5 (gray bar) or another related BMP (black bar). Cleavage mutants produce non-processed BMP5 peptides that bind other non-processed BMP5 peptides to form inactive homodimers or bind and sequester wild-type BMP5 or other related BMPs in defective heterodimers.

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