Isolation and Identification of C‐Mannosylated Hsc70 Conjugates in RAW 264.7 Macrophage‐Like Cells

Abstract

Post‐translational addition of carbohydrate molecules is often necessary for proteins to be fully active. This process, known as glycosylation, usually involves a carbon‐nitrogen or carbon‐oxygen bond. There is, however, a form of glycosylation that involves formation of a carbon‐carbon bond. C‐linked glycosylation in higher organisms consists of the addition of α‐mannopyranoside from dolichyl‐phosphate mannose onto the C‐2 carbon of tryptophan's indole functional group, generating a residue known as C‐mannosyltryptophan. Though the function of this modification is not well characterized, the addition of α‐mannopyranoside to tryptophan residues is thought to play an important role in the innate immune response. (Muroi et al.; Hartmann and Hofsteenge; Hofsteenge et al.)It is known that the consensus sequence for C‐mannosylation is Trp‐x‐x‐Trp/Cys (x could be any amino acid), in which the first tryptophan is modified. (Furmanek and Hosteenge) This canonical sequence has been found in several signaling molecules such as cytokines and cytokine receptors.It has been shown that Hsc70 interacts with proteins containing the C‐mannosyltryptophan residue. With this in mind, Hsc70 pulldowns were done by the Rakus Lab in an attempt to capture some of these conjugates. Several proteins were identified using this procedure, and mass spectrometry showed a mass shift of 162 Da on tryptophan containing peptides – indicative of a C‐mannosyltryptophan residue.One of these modified peptides was identified as belonging to a Type I cytokine receptor – Interleukin 12 Receptor β2 (IL12Rβ2). Notably, one of its target molecules, interleukin‐12 (IL12), also contains the canonical sequence and has been shown to contain a C‐mannosyltryptophan residue. (Doucey et al.)Anti‐ IL12Rβ2 antibodies were used in an attempt to isolate IL12Rβ2 directly. The same was done for a similar protein IL12Rβ1. While IL12Rβ2 appeared to be present in western blots, mass spec data failed to confirm this. IL12Rβ1 was not seen in western blots or mass spec. Upon successful isolation, a more thorough mass spec analysis can confirm or deny the existence of C‐mannosyltryptophan residues in these receptors.Support or Funding InformationMarshall University Chemistry Department; NSF (OIA‐1738707)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.