ABSTRACT
SCUBE2 [signal peptide, CUB domain, EGF (epidermal growth factor)-like protein 2] belongs to an evolutionarily conserved SCUBE protein family, which possesses domain organization characteristic of an N-terminal signal peptide sequence followed by nine EGF-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C-terminus. Despite several genetic analyses suggesting that the zebrafish orthologue of the mammalian SCUBE2 gene participates in HH (Hedgehog) signalling, the complete full-length cDNA and biochemical function for mammalian SCUBE2 on HH signalling remains uninvestigated. In the present study, we isolated the full-length cDNA and studied the role of human SCUBE2 in the HH signalling cascade. When overexpressed, recombinant human SCUBE2 manifests as a secreted surface-anchored glycoprotein. Deletion mapping analysis defines the critical role of the spacer region and/or cysteine-rich repeats for membrane association. Further biochemical analyses and functional reporter assays demonstrated that human SCUBE2 can specifically interact with SHH (Sonic Hedgehog) and SHH receptor PTCH1 (Patched-1), and enhance the SHH signalling activity within the cholesterol-rich raft microdomains of the plasma membranes. Together, our results reveal that human SCUBE2 is a novel positive component of the HH signal, acting upstream of ligand binding at the plasma membrane. Thus human SCUBE2 could play important roles in HH-related biology and pathology, such as during organ development and tumour progression.
Subject(s)
Membrane Glycoproteins/isolation & purification , Membrane Glycoproteins/metabolism , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Calcium-Binding Proteins , Caveolin 1/metabolism , DNA, Complementary/isolation & purification , Extracellular Matrix Proteins/chemistry , Glycosylation , Hedgehog Proteins/metabolism , Humans , Membrane Microdomains/metabolism , Membrane Proteins/chemistry , Mice , Molecular Sequence Data , NIH 3T3 Cells , Open Reading Frames/genetics , Patched Receptors , Patched-1 Receptor , Protein Binding , Protein Structure, Tertiary , Protein Transport , Receptors, Cell Surface/metabolism , Repetitive Sequences, Amino Acid , Sequence Alignment , Signal Transduction , Zebrafish Proteins/chemistryABSTRACT
To determine whether lotus plumule supplementation alleviates acute systemic inflammation in vivo, the BALB/c mice were continuously supplemented with lotus plumule for 3 weeks, following administration with an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) at a concentration of 10mg/kg body weight (BW) to induce acute systemic inflammation. At 24h after injection of LPS, the mice were sacrificed and the visceral organ weight and splenocyte responses were measured. The results showed that lotus plumule supplementation did not significantly affect body weights and IL-6 secretion of splenocyte cultures from BALB/c mice. LPS challenge significantly increased the relative organ weights of the lungs, liver, and spleen, however low dose supplementation (40 mg/kg BW/day) with lotus plumule significantly decreased the relative organ weights of the inflammatory liver, spleen and kidney. Low dose supplementation with lotus plumule significantly increased IL-10 production of splenocyte cultures, however high dose supplementation (800 mg/kg BW/day) significantly decreased IL-10 production. These results suggest that low dose and 3-week supplementation of lotus plumule might alleviate acute systemic inflammation in vivo via decreasing the visceral organ inflammation and increasing the production of anti-inflammatory cytokine IL-10 from splenocytes. These results are valuable for developing future nutraceuticals and anti-inflammatory agents from traditional medicinal foods.