Structure and Stability of C19orf10 as Assessed by Circular Dichroism

Human C19orf10 is a poorly characterized 142‐residue protein that is present in numerous tissues and widely expressed in cultured cells. Homologues of this protein are encoded in organisms ranging from humans to slime molds. C19orf10 and its homologues contain an N‐terminal signal sequence to direct the protein into the endoplasmic reticulum (ER) and a KxEL sequence at the C‐terminus that may result in its retention in the ER. A recent publication (Korf‐Klingebiel et al. , Nature Medicine 21, 140–149, 2015) provided evidence that C19orf10 in monocytes is involved in tissue repair and promotion of angiogenesis after myocardial infarction. Although this suggests that C19orf10 acts as a paracrine signaling factor after secretion, the conserved KxEL sequence suggests functions in the ER that have yet to be investigated. The structure of C19orf10 is not known nor does its sequence suggest any known structural motif. We sought to characterize the C19orf10 structure using circular dichroism (CD) spectroscopy. Recombinant human C19orf10 fused to a C‐terminal poly‐histidine tag was produced as a secreted protein from baculovirus‐infected insect cells. The purified protein was soluble in dilute acetic acid (pH 4.5) as well as in phosphate‐buffered dilute saline (pH 7.5). Spectra at 56°C and below were nearly identical under both buffer conditions and were dominated by an increase in molar ellipticity from 217 nm down to 202 nm with a small negative signal above 217 nm, suggestive of the presence of β‐strands and β‐turns. At 70°C in neutral buffer, the positive signal was replaced by a broad negative peak centered at ~217 nm that did not recover on return to lower temperature. Under acidic conditions the positive signal also became negative with an inflection near 220 nm but continuing downward with decreasing wavelength; however, the spectra recovered upon return to lower temperature. Although the spectra at the two pHs were not identical at high temperature, these results were suggestive of dramatic temperature‐induced changes in structure under both conditions. This data provides evidence that (1) C19orf10 has a global fold that is stable over a pH range from 4.5 to 7.5 and (2) the fold can be regenerated only at acidic pH after heat denaturation. This characterization of recombinant C19orf10 secreted using the quality control mechanism for correct folding in the ER of eukaryotic cells should facilitate further structural studies by serving as a guide to optimize and validate refolding protocols for C19orf10 produced in more robust bacterial expression systems.