Fig. 10

Genomic organization, protein domains, and predicted protein domain structure of SCUBE3. A–B Graphic display of the genomic organization of SCUBE3 and the protein domain structure of its encoded product. Impacts of the disease-causing variants on protein coding are indicated. Our team first reported and characterized eight bi-allelic inactivating variants in SCUBE3 from 18 affected individuals of nine unrelated families who showed a similar phenotype of distinctive craniofacial appearance, dental anomalies, skeletal features, and reduced growth (closed red circles) [21]. Another team then identified recessive mutations of SCUBE3 from a patient with undiagnosed skeletal disease (open red circles) [164]. Two ENU-induced mutant alleles in Scube3 associated with spine deformity including severe kyphosis and kinked tail [164], as well as skeletal abnormalities and altered bone metabolism [165] are also shown (closed black triangles above the diagram) (A). Genomic location of the disease-causing variants of SCUBE3. C.2599 + 2T > C is predicted to result in multiple transcripts with abnormal processing, with p.Asn801Thrfs*127 representing the most common out-of-frame product [21]. Each exon is shown with size to scale, but introns are not. Also, the colors were used to coordinate specific protein domains and their coding exons. For instance, the signal peptide sequence and corresponding exon 1 are both shown in grey. In addition, exons 19 and 20, and the encoded the CUB domain are shown in purple (B). C–D Residues C97 and G204 of human SCUBE3 from AlphaFold2 model. Three disulfide bonds between six cysteine side chains are shown in ‘stick-mode’ in the second EGF-like domain of human SCUBE3; for instance, C97 (in cyan) connects with C110. These six cysteine residues are crucial for disulfide connectivity in structure folding and stability (C). G204 (in green) located on the fifth EGF-like domain is in contact with F101 of the second EGF-like domain and residues M130, M131, G132 and S133 of the third EGF-like domain (atoms colored with carbon in yellow, oxygen in red and nitrogen in blue) in this model. The substitution of residue G204 into D204 may perturb the interactions between the three EGF-like modules in human SCUBE3 (D). E The position of residue I815 in the CUB domain of human SCUBE3. Ribbon model represents human SCUBE3 CUB domain (C804 to Y916) from AlphaFold2 prediction (AF-Q8IX30-F1-model_v3) [187] with the β-strands numbered (in white) based on the bovine spermadhesin CUB domain [224]. I815 is shown in green, and residues within a distance of 5 Å are in yellow. Side chains of these residues buried in the hydrophobic core are shown in ‘stick-mode’ with oxygen atoms in red and nitrogen atoms in blue. Of note, I815 and surrounding residues, including I896, W832, L808, I834, Y913, I844, I911 and F909, form a cluster of buried hydrophobic amino acids in the core of the domain. Replacement of I815 with a polar threonine residue is predicted to destabilize the structure of the CUB domain