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[Frontiers in Bioscience 16, 1505-1516, January 1, 2011] Biological properties of the PrP-like Shadoo protein Nathalie Daude, David Westaway Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada TABLE OF CONTENTS 1. Abstract 2. Introduction 3. SPRN gene expression 3.1. Expression of SPRN mRNA 3.1.1. Mice 3.1.2. Sheep 3.1.3. Cattle 3.1.4. Humans 3.2. Synthesis, maturation and structure of the Shadoo protein 3.2.1. Studies in silico 3.2.2. Shadoo in vitro 4. Shadoo and cellular physiology 4.1. Pathways and binding partners 4.2. Neuroanatomical expression and function 5. Shadoo and the pathogenesis of prion disease 5.1. Down-regulation of Shadoo protein in prion-infected mice 5.2. Shadoo and scrapie disease of sheep 5.3. Germline SPRN mutations and human prion disease 6. Perspective 7. Acknowledgments 8. References 1. ABSTRACT 2. INTRODUCTION The SPRN gene encodes the Shadoo glycoprotein (Sho), a The world was introduced to the cellular prion central nervous system-expressed member of the prion protein PrPC and its transformation to the disease associated protein superfamily. Sho has similarity to two features prion protein PrPSc over twenty-five years ago. While within PrPC's natively unstructured N-terminus, a thousands of publications have since accrued on the hydrophobic domain and tandem repeats with positively properties of these proteins, deciphering physiological charged residues. Indeed, scrutiny of Sho’s biochemical function and how this process or processes are nudged properties in uninfected cells has revealed overlaps with the towards pathophysiological states have proven challenging. properties of PrPC, these including shared protein binding This situation may reflect, in greater part, the observation partners. SPRN is conserved in mammals, as is the prion that PrPC did not belong to a pre-existing protein family of gene PRNP, but in sheep SPRN and PRNP are both marked defined activity. The Doppel protein (Dpl) was identified in by polymorphic variation, suggestive of a shared selection 1999 (1) but, since serving a crucial role in cells of the pressure within these scrapie disease-prone livestock male reproductive tract, did not point to functions that animals. In rodent models of prion disease there are relate to the predominant sites of expression of PrPC such reduced levels of Sho in infected tissues, defining a form of as the central nervous system (CNS), heart and lung. It is cross-regulation between full-length Sho holoprotein and with this backdrop that a new gene denoted SPRN has PrPSc. In human prion disease an SPRN signal peptide emerged onto the scene. SPRN was identified in 2003 and polymorphism is associated with risk for sporadic codes for the protein Shadoo (Sho). Sho has been Creutzfeldt-Jakob Disease (CJD), while two patients with identified as a new CNS-expressed member of the prion early-onset variant CJD carried putatively inactive SPRN protein superfamily. This protein, the focus of this review, alleles. Further investigation of Sho as a novel tracer or comprises the third PrPC-like protein identified thus far in modifier for the accumulation of pathologic forms of PrP higher mammals. While PrP and the testis-expressed Dpl may prove advantageous. protein lie immediately adjacent on chromosome 2, Sho 1505 Prp-like Shadoo protein Figure 1. Domain structures of PrP, Doppel and Shadoo proteins. The schematic in PrP includes octarepeats, hydrophobic region (“HD” in diagonal stripes; α, alpha helices; ß-strands (“ß”) and N-glycosylation sites (CHO). In doppel helix B is not contiguous and has a kink in the middle hence the two parts are labeled B and B’. For Sho the arginine-rich repeats (blue rectangles), HD and the single N-glycosylation site are shown. Regions and attachment positions are approximately to scale. Dashed arrow represent the size of the full-length proteins in amino acids (aa). Open boxes represent a charged region present at the mature amino terminus of each protein. resides on chromosome 7 of mice (2, 3). As discussed in expression in the cell body of pyramidal cells and Purkinje greater depth below, while Dpl has secondary and tertiary cells in the hippocampus and cerebellum, respectively (4); structure similarity to PrP's alpha-helical domain, Sho bears findings were extended by immuno-histochemistry with similarity to two features within PrP's natively unstructured polyclonal antisera directed against Sho peptide sequences N-terminus: these are the hydrophobic domain (HD, (see below). However, Sho expression is also present in referred to by some researchers as the central region), and a other area of the brain including the cerebral cortex, series of tandem repeats with positively charged residues thalamus, and the medulla (4). Bioinformatic analyses of (Figure 1). expressed sequence tags and SAGE libraries have lead to a tempering of the original contention that this gene is mainly 3. SPRN GENE EXPRESSION expressed in neurons (5), suggesting systemic expression in a number of tissues including colon, oesophagus, T-cells, 3.1. Expression of SPRN mRNA muscle and testis, amongst others. Consolidation of these 3.1.1. Mice analyses by analysis of protein expression is an important As in the case of PrP, the entire open reading next step. frame (ORF) of Sho is encoded within one coding exon. Analysis by RT-PCR has shown that Sprn mRNA is mostly 3.1.2. Sheep expressed in brain (3). In situ hybridization in mouse shows Within the CNS, interrelationships between that Sprn is expressed in the adult CNS, with a strong PRNP and SPRN expression have been examined by 1506 Prp-like Shadoo protein quantitative RT-PCR (6), using tissues from both infected http://cgap.nci.nih.gov , http://www.cleanex.isb-sib.ch/, and healthy (normal brain homogenate inoculated) animals. http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly) PRNP and SPRN were found to be expressed “in step”, indicates that human SPRN is transcribed in the adult with the highest expression in the frontal cortex, followed hippocampus, cerebellum, eye and spinal cord. by the cerebellum, the obex and finally the thalamus. Systemically, representation in EST and SAGE databases is However, another study reported no significant difference in consistent with mRNA expression in kidney, liver, lung, relative SPRN mRNA expression between sheep cerebrum and pancreas and prostate. cerebellum in healthy animals, again as assessed by RT-PCR, but did report a positive correlation between relative PRNP 3.2. Synthesis, maturation and structure of the Shadoo and SPRN expression in these two brain regions (7). While the protein absolute expression levels were not reported by Lampo et al. 3.2.1. Studies in silico (7), in the Gossner et al. studies SPRN was expressed 100 to Based upon bioinformatic analysis, Sho was 1000 fold less than PRNP, depending of neuroanatomical predicted to be synthesized in the secretory pathway with region examined (6). These numbers are at apparent variance biochemical features of the protein including an N-terminal with the situation in the rodent CNS, where Sho protein is signal peptide, a series of arginine containing tetrarepeats, a readily detected by the same standard western blotting central HD with strong homology to PrP, a less conserved procedures used for PrPC (i.e. without recourse to the use of C-terminal domain with one consensus N-linked ultrasensitive reagents): (4). While species-specific differences glycosylation site, and lastly, a signal peptide for could of course apply, since the RT-PCR data from sheep were attachment of a GPI anchor (3). The last of these features derived from the use of only a single primer pair (in both were first demonstrated in vitro for one isoform of studies), and since the 2.5 kb sheep SPRN mRNA has a high zebrafish Shadoo (SHO2) by transfection of a FLAG- G+C content (overall figure for the mRNA is 69% GC, and tagged allele into N2a cells followed by PIPLC digestion 78% GC for the ORF region alone) that may confound PCR (10). Most of the other predictions have now been amplification steps, it may be useful to verify the inference of validated with regards to mammalian Sho, as discussed low SPRN mRNA abundance relative to PRNP by other types below. of procedures (e.g., quantitative Northern blot analysis, RNA protection assay). Also these findings are at apparent variance In terms of structural features deduced from with immunohistochemical studies (below, (8)). primary structure, application of contemporary structural prediction algorithms (some capable of accurately Lampo and co-workers have identified genetic predicting the three helices in the NMR structure of the variations in the putative promoter region of the sheep globular domain of PrP, in contradistinction to early studies SPRN gene (8). Five nucleotide substitutions have been which arrived at a 4-helix model (11)) has revealed little of found, as well as one insertion and one deletion. Three interest, other than the puzzling observation that the HD bases changes and one deletion were defined within the region is predicted as a helix (12). The sequence of PrP gene intron and eight nucleotide changes have been found analyzed by the same means yields the same result, with in the 3’UTR. The ultimate significance of these changes the added corollary that transmembrane (TM) forms of PrP remains to be established. Thus far, four natural promoter can be identified in cell-free systems and in cell lysates in region polymorphisms have been assessed by linking them the instance of the A117V Gerstmann–Straussler– to a promoter-deficient firefly luciferase reporter plasmid Scheinker (GSS) mutation (13, 14). The significance of and performing transfections into human SH-SY5Y this finding for Sho remains to be explored but in this neuroblastoma cells.

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