mutations in 15-hydroxyprostaglandin dehydrogenase cause … · 2010-06-10 · schamroth sign in 12...
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Mutations in 15-hydroxyprostaglandin dehydrogenase cause primary hypertrophic osteoarthropathy
Sandeep Uppal1, Christine P Diggle1, Ian M Carr1, Colin W G Fishwick2, Mushtaq Ahmed3, Gamal H. Ibrahim4, Philip S Helliwell1, Anna Latos-
Bieleńska5, Simon E V Phillips6, Alexander F Markham1, Christopher P Bennett3 & David T Bonthron1,3
1Leeds Institute of Molecular Medicine, University of Leeds, St. James’s Hospital, Leeds, U.K.; 2School of Chemistry, University of Leeds; 3Yorkshire
Regional Genetics Service; 4Department of Rheumatology, St. Luke’s Hospital, Bradford; 5Department of Medical Genetics, University of Medical
Sciences, Poznań, Poland; 6Astbury Centre for Structural Molecular Biology, Institute for Molecular and Cellular Biology, University of Leeds
Supplementary Note
Structural modelling of HPGD and the predicted effect of the A140P mutation
To assess the likely effect of the A140P mutation on HPGD function, we constructed a molecular model for the interaction of prostaglandin E2 with the
enzyme, using a crystal structure of human HPGD complexed to NAD, from which the prostaglandin substrate is absent. (See Methods, Figure 3 and
Supplementary Figure 2a-c.)
In our most favourable molecular model, the PGE2 structure is located within a deep hydrophobic tunnel leading from the outside of the protein into
the cavity containing the NAD cofactor (Figure 3a). The 15-OH-containing side-chain of PGE2 lies deep within this pocket, making favourable
hydrophobic interactions with residues L139, V145, F185, L191, I194, and I210, which line this tunnel (Supplementary Figure 2a,b). In addition to
these hydrophobic interactions, the side-chain 15-OH group of PGE2 is predicted to make a hydrogen bond to serine S138, which is also H-bonded to
the backbone amide N-H of alanine A140 (Figure 3b). Our modelling of the A140P mutant predicts that PGE2 binds very poorly within the reaction
cavity of this mutant enzyme. This appears to be a direct consequence of the location of the bulk of the pyrrolidine ring of P140 within a space that in
the wild-type complex is occupied by the PGE2 side-chain 15-OH (Figure 3c). This not only results in considerable steric repulsion between the
hydroxyl and prolyl moieties, but also removes the hydrogen bond that was present in the wild-type structure between A140 and S138. This results in
the side-chain of S138 moving by almost one Ångström, with a consequent loss of hydrogen bonding to the PGE2 15-OH group (Figure 3d). The
combination of steric repulsion and disruption of key hydrogen bonds is therefore predicted to result in loss of catalytic activity in the A140P mutant.
The overall orientation of PGE2 in our docking model is broadly similar to that reported previously from studies of an HPGD homology model, which
also features a hydrogen bond between the PGE2 side-chain hydroxyl and serine S13829. However, a difference between the previous homology model-
based calculations and our present docking studies (which utilise the actual HPGD crystal structure) is that, in the earlier study, in addition to S138,
glutamine residue Q148 was also predicted to make an H-bond contact to the PGE2 side-chain hydroxyl. Indeed, mutagenesis studies underline the
importance of Q148 in maintaining the catalytic efficiency of HPGD29. However, inspection of the HPGD crystal structure reveals that S138 and Q148
are over six Ångströms apart and would be unable to simultaneously form H-bonds to the PGE2 hydroxyl (although it is possible that H-bonding to
Q148 may take place during movement of the PGE2 within the reaction cavity during the oxidation reaction). Alternatively, the important role of Q148
in maintaining the catalytic activity of the enzyme may reflect the fact that Q148 is part of an extensive H-bond network lining the reaction cavity,
involving N95, S193, I190, and T188, which also makes an H-bond contact to a phosphate oxygen of the NAD cofactor (Supplementary Figure 2c).
Figure S3
Supplementary Figure 1
Conservation of the mutated HPGD residue A140. Eight representative vertebrate sequences are aligned, with alanine 140 (A140) coloured pink.
Note that the three bony fish sequences, from Fugu rubripes (puffer fish) Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) are as
divergent from one another as from the five tetrapods. Residues matching the consensus sequence are dark blue, and non-identical residues with a
positive Blosum62 score with the consensus residue are light blue.
Supplementary Figure 2
Molecular model of PGE2 binding to HPGD.
(a) Cross-section through the hydrophobic
tunnel accommodating the 15-OH-bearing
side-chain of PGE2. The surface is colour
coded, such that green represents hydrophobic
regions, and red or blue polar regions.
(b) View from the outside of the
HGPD protein, looking into the
hydrophobic tunnel containing PGE2.
(c) Part of the HPGD crystal structure,
showing that glutamine Q148 is part of a
network of hydrogen bonds lining the reaction
cavity.
Supplementary Figure 3
Mild, progressive clubbing in HPGD heterozygotes. The
Schamroth sign in 12 year-old individual C2 (heterozygous for
the exon 3 frameshift mutation V78QfsX11) reveals ongoing
obliteration of the nailbed angle. In his 38 year-old heterozygous
father (C3) this has progressed to frank clubbing (although still
asymptomatic). In 22 year-old individual A3 (heterozygous for
the A140P mutation) there is a normal nailbed angle, but his 49
year-old heterozygous father (A7) again has developed clear
evidence of clubbing.
.
C2 (12y)
C3 (38y)
A3 (22y)
A7 (49y)
Supplementary Table 1
Patient A1 A2 A4 A9 A10 A13 B1 B2 C1 C4 D1 D2 D3
Current age (years) 21 27 34 17 14 39 14 10 14 35 21 20 13
Skeletal:
Clubbing of fingers and toes + + + + + + + + + + + + +
Diaphyseal expansion/periostosis + + + * * * + * * + + + +
Acro-osteolysis + + + * * * + * * + + + +
Arthralgia of large joints + + + - + - + + + + + + -
Knee-joint effusions + - - - + - + - + + - - -
Skin:
Hyperhidrosis + + + + + - + + + +§ + + +
Pachydermia + + + - - - + - + + + + +
Seborrhoea + + - + + + + - + + + + -
Acne + - + + - - - - + - - - -
Flushing - - - - - - + - + + - - -
Developmental:
Patent ductus arteriosus† - 40,s - - 40,s - 29,s - 34,m - - - -
Delayed cranial suture closure + - - - - - + + + - + + +
* Radiographs not performed § Cervical sympathectomy performed † Gestation (weeks), s=surgically, m=medically treated
Supplementary Table 2
PCR primers used for mutation analysis and expression of HPGD
Mutation analysis
Exon Forward Reverse Amplicon size (bp)
1 dGCTGGCTTGACAGTTTCCTC dCAGCCTCAGCTTCAGCAAAT 381
2 dTTGCTGAAGCTGAGGCTGT dTCTTGCCTTTCTTTCGGTTT 487
3 dTCCACAAACCACACATTGAGA dCCAGCTTTCTGTAACTTCCCTTT 412
4 dTAGGCAAACCCAAAGAATCC dCACATGGGAGCAGAGACATC 652
5 dCCTGGGGAGGCAGAAAAA dTTTATTTGGTTCTTTATGTGATCTGA 514
6 dTGCAGAGTTCAGTAGATAAGAGAAGC dTGCTTGGAATTTAGGCAGAGA 746
7 dTTGGAAGTAGCAATAGTTTAATGACA dTCACCAAGTGCATGAAGGAA 403
HPGD cDNA cassette for bacterial expression
dCCCAGCAGTGGCTGCCATATGCACGTGAACGGC dAATGAAGATAGGATCCGAATATAAGCTATTT
Mutagenic primers for the A140P mutation
dAATATGTCATCTTTACCAGGACTCATGCCCG dCGGGCATGAGTCCTGGTAAAGATGACATATT