biomolecules Article Highly Conserved C-Terminal Region of Indian Hedgehog N-Fragment Contributes to Its Auto-Processing and Multimer Formation Xiaoqing Wang 1,2 , Hao Liu 3 , Yanfang Liu 2, Gefei Han 2, Yushu Wang 2 , Haifeng Chen 3,*, Lin He 2,* and Gang Ma 1,2,* 1 Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] 2 Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] (Y.L.); [email protected] (G.H.); [email protected] (Y.W.) 3 State Key Laboratory of Microbial Metabolism, Department of Bioinformatics and Biostatistics, National Experimental Teaching Center for Life Sciences and Biotechnology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] * Correspondence: [email protected] (H.C.); [email protected] (L.H.); [email protected] (G.M.) Abstract: Hedgehog (HH) is a highly conserved secretory signalling protein family mainly involved in embryonic development, homeostasis, and tumorigenesis. HH is generally synthesised as a precursor, which subsequently undergoes autoproteolytic cleavage to generate an amino-terminal fragment (HH-N), mediating signalling, and a carboxyl-terminal fragment (HH-C), catalysing the Citation: Wang, X.; Liu, H.; Liu, Y.; auto-processing reaction. The N-terminal region of HH-N is required for HH multimer formation Han, G.; Wang, Y.; Chen, H.; He, L.; to promote signal transduction, whilst the functions of the C-terminal region of HH-N remain Ma, G. Highly Conserved C-Terminal ambiguous. This study focused on Indian Hedgehog (IHH), a member of the HH family, to explore Region of Indian Hedgehog the functions of the C-terminal region of the amino-terminal fragment of IHH (IHH-N) via protein N-Fragment Contributes to Its truncation, cell-based assays, and 3D structure prediction. The results revealed that three amino Auto-Processing and Multimer Formation. Biomolecules 2021, 11, 792. acids, including S195, A196, and A197, were crucial for the multimer formation by inserting the https://doi.org/10.3390/ mutual binding of IHH-N proteins. K191, S192, E193, and H194 had an extremely remarkable effect biom11060792 on IHH self-cleavage. In addition, A198, K199, and T200 evidently affected the stability of IHH-N. This work suggested that the C-terminus of IHH-N played an important role in the physiological Academic Editor: Gregory M. Kelly function of IHH at multiple levels, thus deepening the understanding of HH biochemical properties. Received: 8 April 2021 Keywords: Indian Hedgehog; C-terminal region; autoproteolytic cleavage; protein stability; multimer Accepted: 18 May 2021 formation Published: 25 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- The Hedgehog (HH) family is well known for its essential role in the normal embry- iations. onic development of invertebrates and vertebrates and the maintenance of adult tissue homeostasis [1,2]. Deregulation of its signalling pathway leads to the destruction of tissue homeostasis and stem cell self-renewal, further resulting in the occurrence of cancer [3,4]. In higher vertebrates, the HH family mainly contains three homologous proteins: Sonic Copyright: © 2021 by the authors. Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH) [5]. Although Licensee MDPI, Basel, Switzerland. their expression patterns and physiological functions are different, their protein processing This article is an open access article and signal transduction exhibit high similarities. distributed under the terms and HH is regularly synthesised as a precursor in the endoplasmic reticulum of the pro- conditions of the Creative Commons ducing cells, and it undergoes autoproteolysis to generate a functional HH-N, mediat- Attribution (CC BY) license (https:// ing signalling, and a HH-C, catalysing the auto-processing reaction [6,7]. A cholesterol creativecommons.org/licenses/by/ molecule attacks the self-cleavage site to promote the self-cleavage reaction, and binds 4.0/). Biomolecules 2021, 11, 792. https://doi.org/10.3390/biom11060792 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 792 2 of 14 to the C-terminus of HH-N after cleavage. Subsequently, palmitic acid attaches to the N-terminal of HH-N to produce a mature signalling ligand, HHNp [8]. With the help of Dispatched, a transmembrane protein, and heparan sulphate proteoglycans (HSPGs), HHNp is released to the extracellular matrix and forms a soluble multimeric complex, s-HHNp, to facilitate movement. After reaching the recipient cells, s-HHNp binds with the receptor Patched (PTC), relieving the inhibition towards Smoothened (SMO) and further activating the downstream transcription factors GLIs, which trigger the expression of target genes, such as Ptch1 and Gli1 [9]. The N-terminus of HH-N is highly conserved in homologous proteins and various species [10]. Moreover, the palmitic acid modification site [8], HSPG binding sites [11], and PTC binding sites [12] are located at the N-terminus. Three regions, including amino acids 27–34, amino acids 35–48, and the palmitate acceptor site C25 in the N-terminus of SHH-N, are necessary for multimer formation [13]. The previous study depicted that G31R in the N-terminus of SHH-N affects protein stability [14]. These results suggest that the conserved N-terminus of HH-N plays an important role in signal transduction. However, the function of the C-terminus of HH-N is poorly studied. By re-analysing the previously reported IHH crystal structure [15], a 3D structural model of the self- interaction of IHH was discovered. It showed that the S195, A196, and A197 located in the C-terminus of IHH-N could bind to the interior of another IHH molecule. Interestingly, a similar intermolecular interaction model was also found in SHH [16], although the function of this intermolecular interaction remains unclear. In addition, by aligning the amino acid sequences of IHH in different species, the homology of S195, A196, and A197 and the adjacent amino acids were almost 100% (Figure S1), which also indicates the potential conserved biological functions of such regions. In this research, the conserved region of the IHH-N C-terminus was studied by analysing different truncations and point mutations to elucidate the functions of the C-terminus. The results showed that three amino acids, namely, S195, A196, and A197, were crucial for IHH-N multimerization. Meanwhile, K191, S192, E193, and H194 had an extremely substantial effect on IHH self-cleavage. A198, K199, and T200 evidently affected the stability of IHH-N. Models of IHH-N intermolecular interaction and the potential mechanism of full-length IHH self- cleavage were also proposed on the basis of 3D structure, which is important for deepening the understanding of IHH biochemical properties. 2. Materials and Methods 2.1. Construction of HH Mutants Human IHH cDNA was cloned and inserted into the pIND (Sp1)-based inducible expression vector (Invitrogen, Carlsbad, CA, USA) with double FLAG-tags were intro- duced into the IHH-N as described previously [15], or the pCMV-based expression vector (Promega, Madison, WI, USA) with double FLAG-tags were introduced into the IHH-C (pCMV-C-2flags). The mutations were introduced through PCR, employing truncation or single-nucleotide substitution primers. The cDNA of human SHH was amplified from the pLNB-SHH plasmid which was provided by Dr. JM Mason from the Gene Therapy Vector Laboratory (Manhasset, NY, USA). Then, the SHH cDNA was mutated and inserted into the pcDNA3.0-6xMyc vector. In fusion expression protein constructs, the cDNA of enhanced green fluorescent protein (EGFP) was cloned from the pEGFP-C3 vector and inserted into the pCMV-C-2flags vector with IHH-C. All constructs were confirmed by Sanger sequencing. The primers used in this study are listed in Table S1. 2.2. Construction of Stable Cell Line and Gel-Filtration Chromatography The WT or SAA truncated (DSAA) plasmid was transiently transfected into the EcR- CHO (ECHO) cells (Invitrogen, Carlsbad, CA, USA) using Lipofectamine 3000 (Invitrogen). Twenty-four hours (24 h) later, the cells were seeded in a six-well plate via limiting dilution method and cultured in the screening medium (G418, 800 ng/µL, Sigma-Aldrich, St. Louis, MO, USA). After 2–3 weeks, the monoclones were picked for cultivation and detection of Biomolecules 2021, 11, 792 3 of 14 IHH-N expression, then the best clone was selected to expand the culture. After filling in a single-cell layer, ponasterone A (5 µg/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added for another 24 h cultivation. The supernatants were collected and concentrated immediately with Amicon Centricon-4 centrifugal filter devices (Millipore, Bedford, MA, USA) before addition of protease inhibitors (Yeasen, Shanghai, China). After automatic chromatography (Superdex 200 column, GE Company, Boston, MA, USA), the collected aliquots were concentrated by trichloroacetic acid and immunoblotted with anti-FLAG antibody (Invitrogen, MA1-91878, 1:1000; Sigma-Aldrich, F3165, 1:4000) and goat anti- mouse secondary antibody (ThermoFisher Scientific, Waltham, MA, USA, 31430, 1:5000). 2.3. IHH Activity Assay in C3H10T1/2 Cells The C3H10T1/2 cells (ATCCs) were seeded in twenty-four-well plates with an appro- priate density and cultured in a growth medium containing DMEM, 10% foetal bovine serum, and 1% penicillin/streptomycin. The culture supernatants of WT or DSAA stable cell line were added into the growth medium and continued to incubate at 37 ◦C for 4–5 days. Subsequently, the C3H10T1/2 cells were washed in cold PBS and the IHH-N activity was measured using an alkaline phosphatase (AKP) substrate kit (Vector labs, Burlingame, CA, USA, SK-5100). All induction assays were performed in triplicate. 2.4. Transfection and Analysis of HH Mutants pIND-based plasmids were transfected into the ECHO cells by using Lipofectamine 3000 to study the expression of IHH protein.