Highly Conserved C-Terminal Region of Indian Hedgehog N-Fragment Contributes to Its Auto-Processing and Multimer Formation

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 introduced 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 (∆SAA) 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 ∆SAA 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. After 24 h, ponasterone A was added to the cell culture medium. Another 24 h later, the cells were lysed using lysis buffer (Beyotime Shanghai, China) with addition of a protease inhibitor. The cell homogenates were cleared by centrifugation, then separated by 12% SDS-PAGE and immunoblotted with anti-FLAG monoclonal antibody. Similarly, the pCMV-based IHH constructs were transiently transfected into ECHO cells and 48 h later, the cells were lysed and detected with the same antibodies. The pcDNA3.0-6xMyc plasmids were transfected into the HEK293T (293T) cells (ATCCs) and detected using anti-SHH (Santa Cruz Biotechnology, sc- 365112, 1:500) and anti-Myc (Invitrogen, 13–2500, 1:500) monoclonal antibodies. The fusion expression plasmids were transfected into the 293T cells, immunoblotted with anti-FLAG monoclonal antibody and anti-EGFP polyclonal antibody (Abcam, Cambridge, MA, USA, ab5450, 1:5000). Anti-GAPDH HRP-conjugated antibody (Proteintech, Rosemont, IL, USA, HRP-60004, 1:5000) was chosen as the internal control for Western blot. The secondary antibodies used in this experiment were goat anti-mouse IgG (H+L) and rabbit anti goat IgG (H+L) (Invitrogen, A27014, 1:2000). The grey value of the protein bands was analysed by Image J. The ratio of IHH-N (or IHH-C) to the total IHH protein, which is composed of full-length protein and IHH-N (or IHH-C), was calculated with the WT as the standard. Statistical analysis of relative expression was calculated by SPSS.

2.5. Prediction of IHH Protein Structure The software I-TASSER server with default parameters was used for all structural predictions [17]. The predicted structures of the mutated IHH-N and SHH-N were based on the existing structure (PDB ID: 3K7G and 3N1R). The predicted structures of IHH-C and SHH-C were aligned to the crystal structure of HH-C protein from Drosophila melanogaster (PDB ID: 1AT0) by using PyMOL software. The raw and optimised Ca root-mean-square deviations (RMSDs) were calculated by PyMOL. The structures of EGFP-IHH fusion proteins with different mutations were predicted on the basis of the structure of the existing EGFP (PDB ID: 2Y0G) and predicted IHH-C.

2.6. Cell Immunoﬂuorescence of IHH Mutants The WT, ∆SAA, KSEH truncation (∆KSEH), and cholesterol modiﬁcation site mutation C203* plasmids were transfected into ECHO cells as described above. Next, 24 h after ponasterone A was added, anti-FLAG monoclonal antibody was incubated at 37 ◦C for 2 h Biomolecules 2021, 11, 792 4 of 14

to stain the IHHNp on the cell membrane surface. Then, cells were ﬁxed with 80% cold acetone for 30 min at 4 ◦C and labelled with goat anti-mouse Alex488 (Invitrogen, A32723, 1:1000) for 2 h at 37 ◦C. A Leica SP8 laser scanning microscope was used for detecting immunoﬂuorescence.

3. Results 3.1. S195, A196, and A197 Are Involved in Multimerization of IHH-N Protein by Maintaining Intermolecular Interactions In the previous research, the crystal structures of human WT and three brachydactyly type A1 (BDA1)-related mutant IHH-N protein domains were resolved to further under- stand the biochemical consequences of these mutations [15]. Re-analysis of the WT IHH-N crystal structure revealed two interaction models between two symmetry-related molecules of IHH-N: one was the C-terminal binding model where the S195, A196, and A197 located in the C-terminus of molecule A were positioned at the zinc binding site of molecule B (Figure1A), and the other was the N-terminal binding model, where the amino terminus (R39, P40, P41, R42, and K43) of molecule A was located at the zinc binding site of molecule B (Figure S2). These two models have been reported in SHH-N [13,16] and the N-terminal interaction model was considered to be required for multimeric complex formation [13]. However, whether C-terminus, especially S195, A196, and A197, is vital for the multimer formation of IHH-N remains unclear. Gel filtration chromatography showed that the ∆SAA had different elution profiles compared with the WT (Figure1B,C), especially a substantially reduced multimer in fractions 10–13 in ∆SAA. However, no apparent change in monomer was found in fractions 17 and 18 (Figure1C). This result indicated that S195, A196, and A197 were crucial for the multimer formation of IHH-N proteins. Given that HH signalling activity is associated with the multimer level, the influence of ∆SAA on HH signalling activity was elucidated using cell differentiation assay in vitro. The C3H10T1/2 cell line is one of the mesenchymal stem cells that could be differentiated into diverse terminal cells under the stimulation of distinct biological molecules. After responding to HH ligands, these cells are differentiated into mature osteoblasts, and express the corresponding marker AKP. The culture supernatants of the WT or ∆SAA stable cell line were collected and added into the medium of C3H10T1/2 cells, then the expression of AKP was detected after 5 days. The results clearly showed that the AKP activity was significantly impaired in ∆SAA (Figure1D), suggesting a significant attenuation of the IHH signalling activity. Therefore, all results indicated that S195, A196, and A197, which were located in the C-terminus of IHH-N, were essential for multimerization to facilitate signalling activity.

3.2. Conserved C-Terminus of IHH-N Fragment Affects Protein Self-Cleavage and Stability The amino acid sequences of different species were aligned to further clarify the potential functions of the IHH-N C-terminus. The results showed that the homology of S195, A196, and A197 and adjacent amino acids were almost 100% (Figure S1A), indicating the potential conserved biological functions of such region. Ten amino acids (KSEHSAAAKT), including S195, A196, and A197, were initially selected to construct different truncations, and the relevant expression of IHH-N and IHH- C was studied (Figure2A). The results showed that the IHH-N and IHH-C fragments were slightly reduced in ∆SAA but remarkably less in SAAAKT truncation (∆SA ... KT) than that in the WT. In addition, neither IHH-N nor IHH-C was identiﬁed in KSEHSAAAKT truncation (∆KS . . . KT, Figure2B,C). BiomoleculesBiomolecules 20212021, 11, 11, x, 792FOR PEER REVIEW 5 5of of 14 14

Figure 1. S195, A196, and A197 are required for multimer formation of IHH-N. (A) The C-terminal binding model of IHH-N Figureshowed 1. thatS195, the A196, S195, and A196, A197 and are A197 required of molecule for multimer A (pictured formation as backbone of IHH-N. trace) (A) were The positionedC-terminal atbinding the zinc model binding of IHH- site of Nmolecule showed Bthat (pictured the S195, with A196, the and molecular A197 of surface molecule area A coloured (pictured by as electrostatic backbone trace) potential). were positioned (B) Schematic at the of zinc WT andbinding SAA sitetruncation. of molecule * truncated B (pictured amino with acids. the molecular (C) The multimeric surface area form coloured of IHH-N by waselectrostatic analysed potential). using gel-filtration (B) Schematic chromatography. of WT and SAAThe truncation. retention was * truncated the total proteinamino acids. collected (C) inThe the multimeric cell culture form supernatants. of IHH-N was The analysed amount of using multimers gel-filtration in fractions chromatog- 10–13 in raphy.∆SAA The were retention significantly was the reduced total protein compared collected with that in the in thecell WT. culture (D) Thesupernatants. results of C3H10T1/2The amount cellof multimers differentiation in fractions showed 10–13 in ΔSAA were significantly reduced compared with that in the WT. (D) The results of C3H10T1/2 cell differentiation that the AKP activity was significantly impaired in ∆SAA, suggesting a remarkable attenuation of the IHH signalling showed that the AKP activity was significantly impaired in ΔSAA, suggesting a remarkable attenuation of the IHH sig- activity. Data are mean ± s.e.m, n = 3, ** p < 0.01. nalling activity. Data are mean ± s.e.m, n = 3, ** p < 0.01. Once self-cleavage occurs, the IHH-N and IHH-C fragments are generated simultane- 3.2. Conserved C-Terminus of IHH-N Fragment Affects Protein Self-Cleavage and Stability ously, which means that the occurrence of self-cleavage could be manifested by detecting eitherThe of amino these twoacid fragments.sequences Whenof different deleting species S195, were A196, aligned and A197, to further the self-cleavage clarify the po- was tentialonly slightlyfunctions affected of the andIHH-N IHH-N C-terminus. proteins The were results still stable. showed The that deletion the homology of six amino of S195, acids A196,(SAAAKT) and A197 exerted and aadjacent modest amino effect onacids the were self-cleavage almost 100% according (Figure to S1A), the partial indicating reduction the potentialin IHH-C conserved protein. However, biological the functions expression of such of IHH-N region. was significantly reduced compared withTen that amino of IHH-C, acids indicating(KSEHSAAAKT), that the lackincluding of A198, S195, K199, A196, and and T200 A197, may were decrease initially protein se- lectedstability to construct and aggravate different protein truncations, degradation. and the Moreover, relevant expression when all 10of IHH-N amino acidsand IHH- were Cdeleted, was studied neither (Figure IHH-N 2A). nor The IHH-C results could showed be detected, that the implying IHH-N and that IHH-C amino acidsfragments K191, wereS192, slightly E193, and reduced H194 werein ΔSAA involved but remarkably in the autoproteolytic less in SAAAKT cleavage truncation reaction of(Δ full-lengthSA…KT) thanIHH that precursor. in the In total,WT. theIn threeaddition, regions neither had effects IHH-N on proteinnor IHH-C self-cleavage was identified or stability in to KSEHSAAAKTvarying degrees. truncation Further research(ΔKS…KT, was Figure conducted 2B,C). on these regions to confirm this effect in detail.Once self-cleavage occurs, the IHH-N and IHH-C fragments are generated simulta- neously, which means that the occurrence of self-cleavage could be manifested by detecting either of these two fragments. When deleting S195, A196, and A197, the self-cleavage was only slightly affected and IHH-N proteins were still stable. The deletion of six amino acids (SAAAKT) exerted a modest effect on the self-cleavage according to the partial reduction in IHH-C protein. However, the expression of IHH-N was significantly reduced compared with that of IHH-C, indicating that the lack of A198, K199, and T200 may decrease protein stability and aggravate protein degradation. Moreover, when all 10 amino acids were deleted, neither IHH-N nor IHH-C could be detected, implying that amino acids K191, S192, E193, and H194 were involved in the autoproteolytic cleavage reaction of full-length IHH precursor. In total, the three regions had effects on protein self-cleavage Biomolecules 2021, 11, x FOR PEER REVIEW 6 of 14

Biomolecules 2021, 11, 792 6 of 14 or stability to varying degrees. Further research was conducted on these regions to confirm this effect in detail.

Figure 2. Conserved C-terminal region of IHH-N affects protein self-cleavage and stability. (A) Schematic of different Figure 2. Conserved C-terminal region of IHH-N affects protein self-cleavage and stability. (A) Schematic of different truncations. * truncated amino acids. (B,C) Western blot and quantitative statistical analysis of the different truncations. truncations. * truncated amino acids. (B,C) Western blot and quantitative statistical analysis of the different truncations. The grey value of the protein bands was analysed by Image J. The ratio of IHH-N (or IHH-C) to the total IHH protein, Thewhich grey is composed value of the of proteinfull-length bands protein was analysedand IHH-N by (or Image IHH- J.C), The was ratio calculated of IHH-N with (or the IHH-C) WT as to the the standard. total IHH Data protein, are whichmean ± is s.e.m, composed n = 3, of** full-lengthp < 0.01, *** protein p < 0.001, and **** IHH-N p < 0.0001. (or IHH-C), The IHH-N was calculated and IHH-C with frag thements WT aswere the slightly standard. reduced Data are in meanΔSAA± ands.e.m, remarkablyn = 3, ** lessp < 0.01,in ΔSA…KT *** p < 0.001, than that **** pin< WT. 0.0001. IHH-N The and IHH-N IHH-C and were IHH-C not fragments detected completely were slightly in Δ reducedKS...KT. in ∆SAA and remarkably less in ∆SA . . . KT than that in WT. IHH-N and IHH-C were not detected completely in ∆KS...KT. 3.3. K191, S192, E193, and H194 Synergistically Affect Self-Cleavage of IHH Precursor 3.3. K191,The results S192, E193,of ΔKS…KT and H194 revealed Synergistically that K191, Affect S192, Self-Cleavage E193, and H194 of IHH may Precursor play a decisive role inThe IHH results precursor of ∆KS self-cleavage.... KT revealed IHH-N that was K191, expectedly S192, E193,absent and and H194 IHH-C may was play sub- a stantiallydecisive role reduced in IHH in the precursor corresponding self-cleavage. KSEH IHH-Ntruncation was (Figure expectedly 3A–C). absent Then, and KSEH IHH-C was replacedwas substantially with AAAA reduced (Figure in 3A) the correspondingto rule out the possibility KSEH truncation that the (Figuredestroyed3A–C). self-cleav- Then, ageKSEH could was be replaced attributed with to AAAAthe drastic (Figure change3A) toin rulethe spatial out the structure possibility after that KSEH the destroyed deletion. Theself-cleavage result clearly could proved be attributed that although to the drasticthe expr changeession of in IHH-C the spatial slightly structure increased, after KSEHit was stilldeletion. far lower The resultthan that clearly of provedWT, indicating that although that self-cleavage the expression was of IHH-Calso considerably slightly increased, inhib- ited.it was Thus, still farself-cleavage lower than failure that of resulted WT, indicating from KSEH that self-cleavagedeficiency but was not also from considerably severe dis- ruptioninhibited. in Thus,protein self-cleavage structure. failure resulted from KSEH deﬁciency but not from severe disruption in protein structure. BiomoleculesBiomolecules 20212021,, 1111,, x 792 FOR PEER REVIEW 77 of of 14 14

FigureFigure 3. 3. K191,K191, S192, S192, E193, E193, and and H194 H194 are are required required for forIHH IHH self-cleavage. self-cleavage. (A) Schematic (A) Schematic of ΔKSEH of ∆KSEH and 4A and constructs. 4A constructs. Red lettersRed letters indicate indicate replaced replaced amino amino acids; acids; * truncated * truncated amino amino acids. acids.(B,C) Western (B,C) Western blot and blot statistica and statisticall analysis analysis showed showed that IHH- that NIHH-N was absent was absent in ΔKSEH in ∆KSEH and 4A and compared 4A compared with WT, with whereas WT, whereas IHH-C IHH-C was remarkably was remarkably reduced, reduced, indicating indicating that IHH that self- IHH cleavage was considerably affected. Data are mean ± s.e.m, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. self-cleavage was considerably affected. Data are mean ± s.e.m, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Subsequently,Subsequently, four-point four-point mutations mutations were were constructed constructed to determine determine which which amino amino acid acid playsplays a a decisive decisive role role in in K191, K191, S192, S192, E193, E193, an andd H194 H194 (Figure (Figure 44A).A). TheThe resultsresults showedshowed thatthat IHH-NIHH-N and and IHH-C IHH-C were presentpresent inin thesethese mutantsmutants (Figure (Figure4B,D) 4B,D) and and all all expression expression levels levels of ofIHH-C IHH-C were were similar similar to thoseto those of theof the WT WT (Figure (Figure4E), suggesting4E), suggesting that KSEHthat KSEH worked worked together to- getherfor IHH for self-cleavage IHH self-cleavage instead instead of being of being only dependent only dependent on a single on a single amino amino acid. Unexpect-acid. Un- expectedly,edly, the IHH-N the IHH-N protein protein was obviously was obviously reduced reduced in K191A in mutationK191A mutation and slightly and reducedslightly reducedin S192A in and S192A E193A and mutations E193A mutations (Figure4B,C), (Figure indicating 4B,C), indicating that these threethat these amino three acids amino may acidsnot only may be not involved only be in involved autoproteolytic in autoproteolytic cleavage but cleavage also in protein but also stability in protein maintenance. stability maintenance.This conclusion This further conclusion explained further the explai phenotypened the phenotype of the complete of the disappearancecomplete disappear- of the anceIHH-N of the fragment IHH-N butfragment a weak but expression a weak expression of the IHH-C of the fragment IHH-C fragment in KSEH truncationin KSEH trun- and cationKSEH/AAAA and KSEH/AAAA replacement replacement assays (Figure assays3B). (Figure 3B). TheThe occurrence occurrence of of self-cleavage self-cleavage typically typically dep dependsends on on the the attack attack of of cholesterol cholesterol on on the the thioesterthioester bond bond at at the the cleavage cleavage site site [18]. [18 Naturally,]. Naturally, the thecleavage cleavage site must site must be properly be properly presentpresent on the on protein the protein surface surface to facilitate to facilitate the attack the attack (Figure (Figure 4F). 4TheF). The3D structures 3D structures of full- of lengthfull-length IHH IHH with with ΔKSEH∆KSEH and and KSEH/AAAA KSEH/AAAA mutations mutations were were predicted predicted using using I-TASSER I-TASSER software,software, which which was was also also used used for for the the followi followingng SHH SHH protein protein predict predictions,ions, to to determine determine whetherwhether KSEH KSEH affects affects the the presentation presentation of of the the self-cleavage self-cleavage site. site. The The structures structures of of IHH-N IHH-N andand SHH-N SHH-N came came from from the the existing existing structure structure (PDB (PDB ID: ID:3K7G 3K7G and and 3N1R). 3N1R). Meanwhile, Meanwhile, the robustnessthe robustness of the of thepredicted predicted human human IHH-C IHH-C and and SHH-C SHH-C structures structures with with the the published published structurestructure of of drosophila drosophila HH-C HH-C [19] [19] (PDB (PDB 1AT0) 1AT0) were were calculated calculated using using PyMOL PyMOL to to ensure ensure thethe reliability reliability of of modelling. modelling. The The raw raw Ca Ca RMSDs RMSDs for for IHH-C IHH-C and and SHH-C SHH-C were were 1.549 1.549 and and 2.5852.585 Å, Å, respectively, whilstwhilst the the optimised optimised Ca Ca RMSDs RMSDs were were 0.453 0.453 and and 0.823 0.823 Å, respectively, Å, respectively,indicating indicating that their that structures their structures were similar were similar (Figure (Figure S3A). S3A). AsAs expected, expected, the the cleavage cleavage site, site, especially especially the the cholesterol-modified cholesterol-modiﬁed amino amino acid acid G202, G202, waswas trapped insideinside thethe spherical spherical IHH-C IHH-C in in∆ KSEHΔKSEH and and KSEH/AAAA KSEH/AAAA mutations mutations(Figure (Figure4F) . 4F).This This ﬁnding finding indicated indicated that that the probabilitythe probability of cholesterol of cholesterol collision collision and self-cleavageand self-cleavage were BiomoleculesBiomolecules 20212021, ,1111, ,x 792 FOR PEER REVIEW 88 of of 14 14

weresubstantially substantially reduced. reduced. Instead, Instead, the cleavage the cleavage site was site normallywas normally presented presented on the on surface the sur- of facethe predictedof the predicted structure structure of WT, of∆ SAA,WT, Δ andSAA, AKT and truncation AKT truncation (∆AKT) (Δ (FigureAKT) (Figure S3B). S3B).

FigureFigure 4. 4. AnalysisAnalysis of of point point mutations mutations in in KSEH KSEH region region and and structural structural simulation simulation of of KSEH KSEH mutations. mutations. ( (AA)) Schematic Schematic of of pointpoint mutations mutations in in KSEH. KSEH. Red Red letters letters indicate indicate mutant mutant amino amino acid. acid. (B (B,C,C) )Western Western blot blot and and statistical statistical analysis analysis showed showed that that IHH-N could be detected in the corresponding point mutant constructs, but it was obviously reduced in K191A mutation IHH-N could be detected in the corresponding point mutant constructs, but it was obviously reduced in K191A mutation and slightly reduced in S192A and E193A mutations, indicating that these amino acids may have a fractional effect on and slightly reduced in S192A and E193A mutations, indicating that these amino acids may have a fractional effect on protein stability. Data are mean ± s.e.m, n = 3, * p < 0.05, *** p < 0.001. (D,E) Western blot and statistical analysis (n = 3) showedprotein stability.that the expression Data are mean levels± ofs.e.m, IHH-Cn = were 3, * p statistica< 0.05, ***lly psimilar< 0.001. to (thoseD,E) Westernof WT, suggesting blot and statistical that KSEH analysis worked (n =to- 3) gethershowed for that IHH the self-cleavage. expression levels (F) Structural of IHH-C simulation were statistically of IHH similar protein to thoseshowed of WT,that suggestingthe C-terminal that region, KSEH worked including together cho- lesterolfor IHH modification self-cleavage. site (F G202) Structural (blue, KSEH; simulation pink, of SAAAKT; IHH protein and showedred, GGCF), that was the C pr-terminalesented on region, the surface including as α cholesterol-helices in WTmodification IHH but covered site G202 and (blue, changed KSEH; to pink, an ap SAAAKT;eriodical coil and in red, KSEH GGCF), truncation was presented and KSEH/AAAA on the surface replacement. as α-helices This in WTfinding IHH indicatedbut covered that and KSEH changed was critical to an aperiodical for the surface coil inpres KSEHentation truncation of the andself-cleav KSEH/AAAAage site on replacement. IHH protein. This finding indicated that KSEH was critical for the surface presentation of the self-cleavage site on IHH protein. In order to further confirm whether KSEH is decisive for the presentation of the cho- lesterolIn modification order to further site, confirmIHH-N was whether directly KSEH replaced is decisive with forEGFP the and presentation the highly of con- the servedcholesterol C-terminus modification was retained site, IHH-N to construct was directly different replaced fusion proteins with EGFP (Figure and S4A). the highly Con- traryconserved to the expectation,C-terminus wasdeleting retained K191, to S192, construct E193, differentand H194 fusion did not proteins affect the (Figure self-cleav- S4A). ageContrary of EGFP-IHH to the expectation, fusion precursor deleting at K191,all, even S192, though E193, the and 10 H194 conservative did not affect amino the acids self- werecleavage deleted of EGFP-IHHsimultaneously fusion (Fig precursorure S4B–D), at all,and even this finding though was the 10identical conservative with that amino in a previousacids were EGFP-SHH deleted simultaneouslyfusion protein study (Figure [20]. S4B–D), The predicted and this st findingructures was of fusion identical proteins with indicatedthat in a that previous IHH-N EGFP-SHH and EGFP presented fusion protein relatively study independent [20]. The and predicted intact spatial structures struc- of tures,fusion with proteins the self-cleavage indicated that site IHH-N exposed and on EGFPthe surface presented all the relatively time (Figure independent S4E), regard- and lessintact of spatialwhether structures, KSEH was with truncated the self-cleavage or even if 10 site amino exposed acids on were the surfaceknocked all out. the This time result(Figure demonstrated S4E), regardless that ofthe whether normal KSEHpresentati wason truncated of the cholesterol or even if modification 10 amino acids site were de- pendedknocked not out. only This on resultthe KSEH demonstrated region but thatalso theon the normal whole presentation spatial structure of the of cholesterol the IHH protein.modification site depended not only on the KSEH region but also on the whole spatial structure of the IHH protein. 3.4. K186, A187, E188, and N189 Also Severely Affect SHH Self-Cleavage by Obstructing the 3.4. K186, A187, E188, and N189 Also Severely Affect SHH Self-Cleavage by Obstructing the PresentationPresentation of of the the Cholesterol Cholesterol Modification Modification Site Site SHH,SHH, another another member member of of the the HH HH family, family, also also has has a a conserved conserved SHH-N SHH-N CC-terminal-terminal sequencesequence (Figure (Figure S1) S1) and and a a highly highly conserved conserved domain domain consisting consisting of of K186, K186, A187, A187, E188, E188, and and N189N189 corresponding corresponding to to K191, K191, S192, S192, E193, E193, and and H H194194 at at the the same same position position in in IHH. IHH. A A KAEN KAEN truncation (ΔKAEN) was constructed (Figure 5A) to find out whether KAEN affected the Biomolecules 2021, 11, 792 9 of 14 Biomolecules 2021, 11, x FOR PEER REVIEW 9 of 14

truncation (∆KAEN) was constructed (Figure5A) to ﬁnd out whether KAEN affected the self-cleavageself-cleavage as as KSEH KSEH did.did. SHH-N almost almost disappeared, disappeared, whilst whilst SHH-C SHH-C decreased decreased to toa cer- a certaintain degree, degree, which which was was similar similar to to that that of of I IHHHH (Figures (Figure5 B,C).5B,C). The The structure structure of of SHH SHH was was subsequentlysubsequently predicted, predicted, and and the the results results revealed revealed that that deleting deleting KAEN KAEN or or replacing replacing KAEN KAEN withwith AAAA AAAA could could similarly similarly disrupt disrupt the the presentation presentation of of the the self-cleavage self-cleavage site site (Figure (Figure5D). 5D). Overall,Overall, these these results results indicated indicated that that even even though though this regionthis region was not was completely not completely conserved con- inserved the HH in family, the HH it family, affected it self-cleavage affected self-cleavage in an analogical in an analogical manner. manner.

FigureFigure 5. 5.K186, K186, A187, A187, E188, E188, and and N189 N189 affect affect SHH SHH self-cleavage. self-cleavage. ((AA)) SchematicSchematic ofof WTWT andand ∆ΔKAENKAEN constructsconstructs ofof SHH. * * truncatedtruncated aminoamino acids.acids. ( (B,,CC)) Western Western blot blot and and statistical statistical analysis analysis showed showed that that SHH-N SHH-N almost almost disappeared, disappeared, and and the the expression of SHH-C decreased remarkably when KAEN was knocked out. Data are mean ± s.e.m, n = 3, * p < 0.05, **** p < expression of SHH-C decreased remarkably when KAEN was knocked out. Data are mean ± s.e.m, n = 3, * p < 0.05, 0.0001. (D) Structural simulation of SHH protein showed that the C-terminal region, including self-cleavage site between **** p < 0.0001. (D) Structural simulation of SHH protein showed that the C-terminal region, including self-cleavage site G200 and C201 (blue, KAEN; pink, SVAAKS; red, GGCF) was presented on the surface as α-helices in WT but covered betweenand changed G200 andto an C201 aperiodical (blue, KAEN; coil in pink, KAEN SVAAKS; truncation red, and GGCF) KAEN/AAAA was presented replacem on theent. surface This finding as α-helices indicated in WT that but the coveredKAEN andregion changed was critical to an aperiodicalfor the surfac coile presentation in KAEN truncation of the cholesterol and KAEN/AAAA modification replacement. site, similar Thisto that ﬁnding of IHH. indicated that the KAEN region was critical for the surface presentation of the cholesterol modiﬁcation site, similar to that of IHH. 3.5. A198, K199, and T200 Evidently Affect the Stability of IHH-N 3.5. A198,Point K199, mutations and T200 and Evidently truncation Affect were the developed Stability of IHH-Nin the AKT region (Figure 6A), and thePoint expression mutations of IHH-N and truncation and IHH-C were were developed characterized in the AKTto further region clarify (Figure the6A), effect and of theA198, expression K199, and of IHH-N T200 on and protein IHH-C stability were characterized and self-cleavage. to further The clarify point the mutations effect of A198,K199A K199,and T200A and T200 showed on protein almost stability similar andexpression self-cleavage. levels of The IHH-N point and mutations IHH-C K199Acompared and to T200Athat of showed WT (Figure almost 6B,C), similar indicating expression that levels mutating of IHH-N a single and amino IHH-C acid compared did not to affect that ofthe WTstability (Figure of6 B,C),IHH-N. indicating However, that when mutating three aamin singleo acids amino were acid deleted did not altogether, affect the the stability expres- ofsion IHH-N. of IHH-C However, was whennormal three but aminothat of acidsIHH-N were remarkably deleted altogether, decreased, the thereby expression revealing of IHH-Cthat A198, was normalK199, and but T200 that ofacted IHH-N as a remarkablycombination decreased, to affect protein thereby stability. revealing that A198, K199, and T200 acted as a combination to affect protein stability. Biomolecules 2021,, 11,, x 792 FOR PEER REVIEW 1010 of of 14 14

Figure 6. A198, K199, and T200 affect the stability of IHH-N. (A) Schematic of point mutations and truncations. Red letters Figure 6. A198, K199, and T200 affect the stability of IHH-N. (A) Schematic of point mutations and truncations. Red letters indicate point-mutation amino acids; * truncated amino acids. (B,C) Western blot and statistical analysis showed that the indicate point-mutation amino acids; * truncated amino acids. (B,C) Western blot and statistical analysis showed that the expression of point mutations were roughly identical to those of WT. After A198, K199, and T200 were deleted, the normal expressionexpression of pointIHH-C mutations revealed werethat the roughly self-cleavage identical was to those not affected, of WT. After whereas A198, the K199, decrease and T200 in IHH-N were deleted, indicated the that normal the stabilityexpression may of be IHH-C affected. revealed Data are that mean the self-cleavage ± s.e.m, n = 4, was * p < not 0.05, affected, ** p < 0.01. whereas the decrease in IHH-N indicated that the stability may be affected. Data are mean ± s.e.m, n = 4, * p < 0.05, ** p < 0.01. 4. Discussion 4. DiscussionIHH is one of the secreted ligands in the HH signalling pathway, and it plays an importantIHH role is one in regulating of the secreted cartilage ligands formation in the and HH development signalling pathway, [5]. Missense and itmutations plays an inimportant IHH lead role to skeletal in regulating developmental cartilage formation defects, such and developmentas BDA1 [21] [and5]. Missense acrocapitofemoral mutations dysplasiain IHH lead (ACFD) to skeletal [22]. The developmental N-terminus defects,of HH-N such plays as a BDA1 vital role [21] in and multimer acrocapitofemoral formation, whilstdysplasia the (ACFD)physiological [22]. ThefunctionsN-terminus of the ofC-terminus HH-N plays of HH-N a vital have role in not multimer been elucidated. formation, whilstThe the structure physiological of SHH-N functions was offirst the reportedC-terminus in 1995 of HH-N [16], and have the not C been-terminus elucidated. of one SHH-NThe (A194 structure and K195) of SHH-N was revealed was first to reported be positioned in 1995 at [16 the], andzinc-binding the C-terminus region ofof onean- otherSHH-N SHH-N. (A194 andThis K195) interaction was revealed might contribute to be positioned to maintaining at the zinc-binding zinc binding region and of anotherthe po- tentialSHH-N. hydrolytic This interaction activity mightof SHH-N contribute [16], alth toough maintaining the hydrolase zinc binding activity and is not the potentialrequired forhydrolytic Hh signalling activity transduction of SHH-N [[23].16], althoughHowever, the zinc hydrolase is indeed activity essential is notfor HH required protein for sta- Hh bilitysignalling and signalling transduction activity [23 ].[23,24]. However, Moreover, zinc is zinc indeed makes essential a vital contribution for HH protein to the stability bind- ingand between signalling HH activity and Hhip, [23,24 which]. Moreover, is a highly zinc conserved makes a vitaland vertebrate-specific contribution to the inhibitor binding ofbetween Hh signalling HH and [25]. Hhip, A whichrecent isstudy a highly revealed conserved that the and cholesteryl vertebrate-specific moiety associated inhibitor of with Hh signalling [25]. A recent study revealed that the cholesteryl moiety associated with seven seven amino acids (191VAAKSGG197) in the C-terminus of SHH-N is embedded in a cavity amino acids ( VAAKSGG ) in the C-terminus of SHH-N is embedded in a cavity of of receptor PTC1191 to relieve th197e inhibition of SMO by PTC1 and stimulate the Hh signalling receptor PTC1 to relieve the inhibition of SMO by PTC1 and stimulate the Hh signalling pathway [26]. All the above studies implied that the C-terminus of HH may exert multiple pathway [26]. All the above studies implied that the C-terminus of HH may exert multiple vital functions to regulate protein stability, partner binding, and signalling activity. vital functions to regulate protein stability, partner binding, and signalling activity. In this study, the highly conserved C-terminus of IHH-N was divided into three re- In this study, the highly conserved C-terminus of IHH-N was divided into three gions to analyse their functions in self-cleavage, protein stability and multimer formation. regions to analyse their functions in self-cleavage, protein stability and multimer formation. The functions of these three regions overlap one another but have their own emphasis. The functions of these three regions overlap one another but have their own emphasis. For For example, K191, S192, E193, and H194 significantly affected the autoproteolytic cleav- example, K191, S192, E193, and H194 significantly affected the autoproteolytic cleavage age reaction by obstructing the presentation of the cholesterol modification site. A198, reaction by obstructing the presentation of the cholesterol modification site. A198, K199, K199,and T200 and mainlyT200 mainly affected affected the stability the stability of IHH-N. of IHH-N. In addition, In addition, inserting inserting S195, S195, A196, A196, and andA197 A197 in another in another adjacent adjacent IHH-N IHH-N molecule molecule contributed contributed to IHH-N to IHH-N multimer multimer formation. formation. Biomolecules 2021, 11, 792 11 of 14

Under physiological conditions, the IHH protein is attacked by a cholesterol molecule to promote the self-cleavage reaction and form two different fragments. The relatively stable IHH-C was normal in the ∆AKT but slightly reduced in the ∆SAA. A more drastic decrease occurred in the ∆KSEH, indicating that the autoproteolytic cleavage reaction was obstructed to a great extent, which demonstrated that the KSEH region was the most critical region for self-cleavage. Similar discoveries were found in SHH. Subsequently, the possible mechanism was clarified through structural prediction and the results showed that the cholesterol modification site could not be normally presented on the protein surface in ∆KSEH, which then reduced the probability of cholesterol colliding with the self-cleavage site and caused the self-cleavage reaction unsuccessfully. However, it cannot be ruled out the possibility that the KSEH deletion or substitution changes the IHH structure, causing self-cleavage to be affected; thus, further study is needed. However, the EGFP replacement assay illustrated that the KSEH region was not decisive to the self-cleavage, and the overall structure of the IHH-N had a considerable effect. In addition, the self-cleavage was slightly affected in ∆SAA and ∆SA ... KT, possibly due to the mild changes of the polypeptide chain surrounding the cholesterol modification site. All results strongly indicate that the presentation of the self-cleavage site was dependent on the KSEH region and 3D structure of the whole IHH-N protein. This mechanism expands the understanding of the factors that influence protein self- cleavage. Two types of factors affecting autoproteolytic cleavage have been proposed prior to this study. One factor is cleavage site mutations, such as C203A, which affect the formation of thioester bonds and prevent self-cleavage from occurring [27]. The other one is HH-C active centre mutations, such as H329Y, which destroy the activity of HH-C as a cholesterol transferase; thus, the HH protein could not be self-cleaved [6]. Another study has shown that mutations or deletions of certain amino acids in the N-terminus of SHH-N also affect the self-cleavage [13], but the reason remains unclear. This, we predicted that these amino acids may also be involved in maintaining the correct presentation of the self-cleavage site or the complete 3D structure of IHH-N. In this research, it was found that ∆AKT did not affect the self-cleavage but significantly affected the protein stability. Unfortunately, the potential mechanism could not be predicted due to the lack of AKT in the existing SHH and IHH protein structures because of AKT hydrolysis in crystallisation [15,16]. In fact, a large number of studies have found that mutations in different regions of the HH-N would reduce protein stability. For example, G31R in the N-terminus of SHH-N [14], BDA1-related mutations in the middle of IHH- N[15], and ∆AKT in the C-terminus of IHH-N in this study. In addition, we also found that ACFD-associated mutation V190A [22], which is closed to the KSEH in the C-terminus of IHH-N, affected the self-cleavage slightly but reduced protein stability obviously (Figure S5), further supporting the significance of the IHH-N C-terminus and also implying that V190A mutation may cause disease in part by reducing the IHH-N protein level. Several studies tried to clarify the potential mechanism of the decrease in protein stability. Traiffort et al. found that the eight mutations on SHH, located near the narrow gap bound to zinc, cause a significant decrease in SHH-N [14]. Three of the eight amino acids are located in the zinc-binding region, indicating that the mutations of these three amino acids affect zinc binding and reduce the stability of SHH-N. Although the other five amino acids are not in this region, reducing the stability by affecting zinc binding indirectly is possible. The other two studies found that E95K and D100E mutations decrease the stability of IHH-N [15] and verified that these two mutations affect calcium binding because they are close to the calcium-binding groove and are involved in the interaction with the receptor and binding partners [28]. Therefore, A198, K199, T200, and even K191, which is also close to the zinc/calcium-binding groove, may affect protein stability by directly or indirectly binding with zinc or calcium or both. However, this hypothesis requires further in-depth research. The HH family consists of secretory signal proteins, whose long-range signalling is mainly controlled by the formation of soluble multimers. Therefore, multimerization Biomolecules 2021, 11, 792 12 of 14

is crucial for the normal biological function of HH proteins. In view of the C-terminal binding model, S195, A196, and A197 promote multimerization by binding to another IHH-N molecule at the zinc-binding site. This study proved that SAA was pivotal for multimer formation and signal activity by using gel-filtration chromatography and cell- based AKP assay. Cholesterol and palmitic acid modifications were identified to be essential for multimer formation. The absence of palmitic acid or cholesterol modification prevents the multimer formation of HH-N proteins [9,29]. In our study, gel-filtration chromatography results showed that a small amount of multimers could still be detected in tubes 13–15 in SAA truncation, indicating that SAA truncation has normal lipid modification. Cell immunofluorescence results also confirmed this point as ∆SAA could be anchored on the cell membrane, similar to WT, as shown in Figure S6 [30,31]. These results implied that the multimer decrease in ∆SAA was not related to lipid deficiency. Based on the 3D structure, the essential function of the SHH-N N-terminus in multimerization [13] has given new indication that not only lipid modifications but also the interaction between two symmetry-related IHH-N affect multimer formation. Our data show that the highly conserved C-terminus of IHH-N was also crucial for multimer formation, thus providing a new standpoint for the study of multimerization. Furthermore, given the published SHH crystal structure [16], A194 and K195 might be involved in SHH multimerization as two key amino acids for the interaction between two symmetry-related SHH-N molecules. IHH is a secreted protein of the HH family that mainly plays an important regula- tory role in cartilage formation and development. IHH is highly conserved in different species, especially the N-terminal signal fragment, which is 98% conserved in mice and humans. The highly conserved C-terminus of IHH-N, which is located near the cleavage and cholesterol-modifying sites, has sparked the research interest of the authors. This study clearly described the important role of the C-terminus in self-cleavage, protein stability, and multimer formation in accordance with the crystal structure of the IHH-N protein, thereby providing an important basis for further elaboration of the mechanism of self-cleavage and multimer formation.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/biom11060792/s1, Figure S1: Conservation of the C-terminal region of HH-N, Figure S2: N-terminal binding model between two molecules of IHH-N, Figure S3: Structural simulation of SAA truncation and AKT truncation proteins, Figure S4: Analysis of EGFP-IHH fusion proteins’ auto-processing, Figure S5: Acrocapitofemoral dysplasia-related mutant V190A affects protein self- cleavage and stability, Figure S6: Cell immunofluorescence of SAA truncation, Table S1: Primers used in this study. Author Contributions: Conceptualization, G.M.; Investigation, X.W., Y.L., and G.H.; Methodology, G.M., X.W., and H.C.; Software, H.L. and H.C.; Validation, X.W., Y.L., and G.H.; Formal Analysis, G.M. and X.W.; Writing—Original Draft Preparation, X.W.; Writing—Review and Editing, Y.W. and G.M.; Supervision, G.M.; Funding Acquisition, L.H. and G.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Nature Science Foundation of China (31970775, 31671504). Informed Consent Statement: Not applicable. Data Availability Statement: All data are contained within the manuscript. Acknowledgments: We thank Congzhao Zhou and Jiang Yu from the University of Science and Technology of China for their helpful discussion about the 3D structure of IHH-N. Conflicts of Interest: The authors declare no conflict of interest. Biomolecules 2021, 11, 792 13 of 14

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