Characterization of CNPY5 and its family members

Abstract

The Canopy (CNPY) family consists of four members predicted to be soluble proteins localized to the endoplasmic reticulum (ER). They are involved in a wide array of processes, including angiogenesis, cell adhesion, and host defense. CNPYs are thought to do so via regulation of secretory transport of a diverse group of proteins, such as immunoglobulin M, growth factor receptors, toll‐like receptors, and the low‐density lipoprotein receptor. Thus far, a comparative analysis of the mammalian CNPY family is missing. Bioinformatic analysis shows that mammalian CNPYs, except the CNPY1 homolog, have N‐terminal signal sequences and C‐terminal ER‐retention signals and that mammals have an additional member CNPY5, also known as plasma cell‐induced ER protein 1/marginal zone B cell‐specific protein 1. Canopy proteins are particularly homologous in four hydrophobic alpha‐helical regions and contain three conserved disulfide bonds. This sequence signature is characteristic for the saposin‐like superfamily and strongly argues that CNPYs share this common saposin fold. We showed that CNPY2, 3, 4, and 5 (termed CNPYs) localize to the ER. In radioactive pulse‐chase experiments, we found that CNPYs rapidly form disulfide bonds and fold within minutes into their native forms. Disulfide bonds in native CNPYs remain sensitive to low concentrations of dithiothreitol (DTT) suggesting that the cysteine residues forming them are relatively accessible to solutes. Possible roles of CNPYs in the folding of secretory proteins in the ER are discussed.

The special unfolded protein response in plasma cells

“…MZB1 was recently shown to belong to the CNPY family of saposin‐like ER‐resident proteins 94 . Unlike the other saposins, which are involved in lipid binding, the five members of the CNPY family bind proteins, with CNPY2‐4 and MZB1 (CNPY5) regulating either folding or secretion/surface trafficking of LDLR, Toll‐like receptors, and Ig chains 94 .…”

“…MZB1 was recently shown to belong to the CNPY family of saposin‐like ER‐resident proteins 94 . Unlike the other saposins, which are involved in lipid binding, the five members of the CNPY family bind proteins, with CNPY2‐4 and MZB1 (CNPY5) regulating either folding or secretion/surface trafficking of LDLR, Toll‐like receptors, and Ig chains 94 . Both CNPY3 and MZB1 have been reported to be co‐chaperones of GRP94, and their functional relationship will be further elucidated now that the crystal structure of MZB1 is available and provides functional insights 94,95 .…”

Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint

“…For immunoprecipitation: we used the previously described polyclonal rabbit anti-gp160 antibody 40336 which recognizes all forms of gp120 (Land et al, 2003), polyclonal antibody HT3 (NIH531) which was obtained from the NIH AIDS reagent program and a-HA tag antibody ''MrBrown'' produced by us (Schildknegt et al, 2019).…”

Intramolecular quality control: HIV-1 Envelope gp160 signal-peptide cleavage as a functional folding checkpoint

“…For immunoprecipitation: we used the previously described polyclonal rabbit anti-gp160 antibody 40336 which recognizes all forms of gp120 (Land et al, 2003), polyclonal antibody HT3 (NIH531) which was obtained from the NIH AIDS reagent program and -HA tag antibody "MrBrown" produced by us (Schildknegt et al, 2019).…”