Supplementary Materials

A novel rare variant R292H in RTN4R affects growth cone formation and

possibly contributes to schizophrenia susceptibility

Hiroki Kimura1, Yuki Fujita2, Takeshi Kawabata3, Kanako Ishizuka1, Chenyao Wang1, Yoshimi Iwayama4, Yuko Okahisa5, Itaru

Kushima1, Mako Morikawa1, Yota Uno1, Takashi Okada1, Masashi Ikeda6, Toshiya Inada1, Aleksic Branko1, Daisuke Mori1, Takeo

Yoshikawa4, Nakao Iwata6, Haruki Nakamura3, Toshihide Yamashita2, Norio Ozaki1

1 Department of Psychiatry, Nagoya University Graduate School of Medicine, Nagoya, Japan

2 Department of Molecular Neuroscience, Osaka University Graduate School of Medicine, Osaka, Japan

3 Laboratory of Protein Informatics Institute for Protein Research, Osaka University Graduate School of medicine, Osaka, Japan 4 Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Wako, Saitama, Japan

5 Department of psychiatry, Okayama University Graduate School of Medicine, Okayama, Japan

6 Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Aichi, Japan

*Corresponding author:

Branko Aleksic, MD, PhD

Associate Professor

Department of Psychiatry, Nagoya University Graduate School of Medicine

65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

Keywords: RTN4R, single nucleotide variant, schizophrenia, next generation sequencing, Nogo

Contents of Supplementary Materials

Supplementary Methods: Modeling of the 3D complex structure of RTN4R and LINGO1

Figure S1: A schematic diagram indicating the procedures used to model the 3D complex structures of RTN4R and LINGO1

Figure S2: Subcellular localization of RTN4R wild type (WT) or mutants in HEK293 cells

Figure S3: Subcellular localization of RTN4R wild type (WT) or mutants in chick retinal neurons

Figure S4: Pedigrees of the subject diagnosed as SCZ with RTN4R-R292H

Supplementary Methods: Modeling of the 3D complex structure of RTN4R and LINGO1

The 3D complex structure of RTN4R and LINGO1 was modeled by superimposing each of their monomeric 3D structures onto the complex 3D structure of their homologs. This procedure is summarized in Figure S1. First, the monomeric crystal structures of human RTN4R (PDBID: 1p8t, chain A) and of human LINGO1

(PDBID: 4oqt, chain A) were prepared. Second, the HOMCOS server1 found the template structure (PDBcode: 4rca) by the condition that the 292-nd site of RTN4R became an interface for other proteins. The complex structure (PDBID: 4rca) is composed of human SLITRK1 (chain B) and the N-terminal domain of PTPRD (chain

A). The sequence of the protein SLITRK1 is similar to that of RTN4R, with 33.5% sequence identity. Importantly, both the protein LINGO1 and PTPRD have an

immunoglobulin (Ig)-like domain, which is in the region 381-477 of LINGO1 and in the region 233-320 of PTPRD. Although their sequence similarities are not high (sequence identity = 19.5%), they have similar 3D structures. Third, we superimposed the structure of RTN4R (4oqt, chain A) on the SLITRK1 structure (4rca, chain B), and superimposed the Ig-like domain of LINGO1 (4oqt, chain A, 381-477) on the Ig-like domain of PTPRD (4rca, chain A, 233-320). The 3D superimpositions were calculated using the program MATRAS2. Combining these two superimposed structures, we

obtained a superimposition model of the 3D complex structure of RTN4R and LINGO1.

This superimposition model was then refined using three programs: MODELLER, UCSF Chimera and myPresto. First, the program MODELLER version 9.163 rebuilt all the atoms by regarding the superimposition model as the template. Next, the “Rotamer” function of UCSF Chimera4 was used to modify the sidechain conformation of the 292-nd site. We chose the rotamer with the highest probability value. Finally, the program cosgene of the program package myPresto version 4.4005 was used to optimize the conformation. We used the implicit solvent model (GB/SA; generalized Born model with solvent accessible surface area term) for this optimization. The mutated structure R292H was modelled in the same way except that the input sequence for MODELLER was mutated with the R292H mutation.

As explained above, the template complex structure was selected so that the 292-nd site of RTN4R was in contact with other proteins. In other words, the model was built to answer the following question: if the 292-nd site of RTN4R does make contact with other proteins, what protein is the most probable protein to interact with RTN4R. Our current answer is that LINGO1 is the most probable to interact with RTN4R. Finally, we note that the reliability of the current structural model is limited, because the sequence similarity of the Ig-like domains of LINGO 1 and PTPRD is not so high as the reliable homologous interaction is provided.

Figure S1: A schematic diagram indicating the procedures used to model the 3D complex structures of RTN4R and LINGO1

Figure S2: Subcellular localization of RTN4R wild type (WT) or mutants in HEK293

cells

Legend: EGFP-fused RTN4R WT or mutant expressing vectors were transfected into HEK293 cells and cultured for 48 h. Scale bar, 100 μm. There were no obvious differences in subcellular localization between RTN4R wild type (WT) and the mutants

Figure S3: Subcellular localization of RTN4R wild type (WT) or mutants in chick

retinal neurons

Legend:

EGFP-fused RTN4R WT or mutant expressing vectors were transfected into chick retinal neurons and cultured for 48 h. Scale bar, 20 μm. There were no obvious differences in subcellular localization between RTN4R wild type (WT) and the mutants

Figure S4: Pedigrees of the SCZ subjects with RTN4R-R292H

Legend: Subjects with RTN4R-R292H were indicated by black arrow. We could get the DNA from the daughter of Subject 2 diagnosed as SCZ, and revealed that she was also RTN4R-R292H carrier.

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