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Fig. 1 | Journal of Biomedical Science

Fig. 1

From: The biology of SCUBE

Fig. 1

Protein domains, phylogenetic analysis, genomic organization, sequence alignment, and sequence identity of SCUBE protein family. A Graphic illustration of the domain structure of human SCUBE1, 2 and 3 (see Table 1). SP, signal peptide sequence; E, EGF-like domain (grey shade indicated the calcium-binding EGF module); Cys-rich, cysteine-rich repeats; CUB, the CUB domain. “Y” marks the potential N-linked glycosylated sites of each SCUBE protein. Protein domain sequence identity shared among human SCUBE members is calculated at the bottom; the highest homology was found in the CUB domain (83%), followed by the EGF-like (73%) and Cys-rich (66%) domains. The spacer region appears to have the lowest homology (34%) and may be associated with the unique functions of each SCUBE member. B Phylogenetic tree of the SCUBE family. Similarity of human (h), mouse (m), zebrafish (z), eagle (e), lizard (l), or frog (f) SCUBE protein sequences was analyzed by using the Lasegene MEGALIGN program (ClustalW algorithm). The length of each pair of branches represents the phylogenic distance between sequence pairs. Below the tree is a scale indicating the number of “Nucleotide Substitutions” for both DNA and protein sequences. C Genomic structure of human SCUBE1. SCUBE1 gene consists of 22 exons spanning about 140 kb on chromosome 22. The exon–intron boundaries are well preserved among the SCUBE gene members, which suggests possible gene duplication during evolution. Of note, SCUBE genomic organizations derived from zebrafish, mouse and human genomes (http://genome.ucsc.edu) follow a modular arrangement, with the signal peptide sequence, each epidermal growth factor (EGF)-like domain, and Cys-rich motif, each encoded by a single exon. In addition, the spacer region is encoded by five exons and the CUB is encoded by two exons. D Amino acid sequence identity of SCUBE family. Data in the upper right (blue shading) represent the sequence identity in EGF-like domain, and data on the bottom left (orange shading) represent the sequence identity in spacer region between SCUBE proteins (upper panel). Data on the upper right (gray shading) present the sequence identity in Cys-rich domain, and data on the bottom left (yellow shading) present the sequence identity in CUB domain between SCUBE proteins (lower panel). E Sequence alignment, sequence identity, and structural comparison of EGF-like repeats of human SCUBE proteins. Protein sequences of SCUBE1, SCUBE2 and SCUBE3 used for alignment were derived from NCBI Reference Sequence Database, as listed in Table 1. Symbols use the one-letter code for amino acids. A dash indicates a gap. The conserved cysteines are numbered 1 to 6, and the predicted disulfide bonds form as follows: C1-C3, C2-C4, and C5-C6. Amino acids indicated below highlight the canonical calcium binding (cb) consensus sequence (D/N)-X-(D/N)-E/Q-Xm (D/N*) Xn (Y/F), where m and n are variable and the asterisk indicates potential β-hydroxylation [17]. Of note, two triplets of cbEGF1-3 or cbEGF7-9 modules (amino acid residue boundaries marked in red fonts) are well conserved in all SCUBE members. F Structural comparison of SCUBE3 second and seventh cbEGF-like domains with human NOTCH1 fifth cbEGF-like domain. 3D models of the second and seventh EGF-like domains of human SCUBE3 obtained from AlphaFold2 (AF-Q8IX30-F1-model_v3) [187] were superimposed on the fifth cbEGF-like domain of human NOTCH1 (PDB 5FM9) [221]. These EGF-like domains contain a short calcium binding motif sequence signature: (D/N)-X-(D/N)-E/Q-Xm-(D/N*)-Xn-(Y/F), where m and n are variable and the asterisk indicates potential β-hydroxylation [17]. This canonical calcium coordination module is found near at the N-terminus. The residues coordinating calcium ion (in green) are shown as sticks, and the coordination as pink dotted lines. The five calcium-binding motif residues indicated in E are presented as cyan sticks; Asn and Phe/Try between C3 and C4 of these EGF-like domains are consistent with the substrate sequence pattern of Asp/Asn-β-hydroxylase [222, 223]. The three paired cysteines (C1:C3, C2:C4, and C5:C6) are presented as yellow sticks. Atoms are colored, with oxygen in red, nitrogen in blue and sulfur in gold. This figure was produced using PyMOL (Schrödinger, LLC. The PyMOL molecular graphics system, version 1.8, https://pymol.org). Nt, NH2 terminus; Ct, COOH terminus

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