Ructure that is highly conserved in mammalian CDH13. It is also close to the residues involved in the recently identified ”Xdimer” dimerization configuration between EC1 and EC2 of CDH13, which is important for homodimerization, adhesion and neurite outgrowth [35]. Thus, R174W might influence these functions of CDH13, as well as its angiogenic effects that are mediated by EC1 and EC5 [49]. CDH13 is a glycoprotein containing several glycosylation sites where N-linked oligosaccharides are attached and promote proper folding in the ER and protein stability [50]. The N39S, A376T,I585V and L643R variants are in close proximity (63 amino acid residues) to N glycosylation sites. However, none of the variants are located at a glycosylation site so it is unlikely that any of them cause misfolding or instability by preventing proper glycosylation. This is also demonstrated by the molecular weight which is the same for all the CDH13 variants studied here. The missense variants A376T, I585V and L643R are found in extracellular domains 2, 4, and 5, respectively. The L643R is located about fifty amino acid residues from the GPI anchor in the mature protein [50]. The functional roles of these residues and protein domains are less clear and since the in silico modelingPLOS ONE | www.plosone.orgCDH13 Coding Variants in ADHDpredicted the mutations to be benign, the lack of clear effects of the missense variants was not unexpected.CDH13 Protein Expression, Intracellular Localization and Function of CDHIn previous studies on human aortic smooth muscle cells CDH13 has been identified as a cell surface expressed, LDLbinding protein of around 130 kDa and 105 kDa [51]. The cell surface expression pattern and LDL-binding properties were also shown in HEK293 cells transfected with CDH13 [52]. Immunostaining of endogenously expressed CDH13 in a human keratinocyte cell line (DJM-1) revealed a band of approximately 105 kDa [18]. In most studies CDH13 has been found at the extracellular surface of the plasma membrane [52,53]. However, expression in other cellular compartments, such as the nucleus and centrosomes in endothelial cells, has also been reported [54].Evolocumab Expression of CDH13 was also observed in neural cytoplasm as well as membrane and neurites in staining of the adult human cerebral cortex [25].Cemdisiran Our findings show that native wild type and variant CDH13 proteins, of approximately 105 kDa (Fig.PMID:24576999 2), are expressed in CHO cells on the cell membrane (Fig. 3). In line with previous findings [16,18] we did not observe increased signal between adjacent cells or CDH13 accumulation at sites of intercellular contacts in CDH13 stained cells, as is commonly observed with classical cadherins such as E-, N-[55] and P-cadherin [18]. The wild type protein and the seven variants showed similar expression levels and localization. In order to detect partial intracellular accumulation of abnormal CDH13, which would be missed by membrane staining, we permeabilised the cells before staining for CDH13. Cytoplasmic localization of membrane proteins may represent abnormal accumulation in the ER which is commonly associated with disease. For instance, in the case of Crohn’s disease-associated SNPs in E-cadherin, a variant was associated with the formation of a truncated E-cadherin, and cytoplasmic accumulation, instead of membrane expression, in the intestinal epithelium of affected patients and in transfected cells [56]. Our findings in CHO cells, however, show no such effects of th.