The Chaperone Activity of Clusterin is Dependent on Glycosylation and Redox Environment

Abstract

Background/Aims: Clusterin (CLU), also known as Apolipoprotein J (ApoJ) is a highly glycosylated extracellular chaperone. In humans it is expressed from a broad spectrum of tissues and related to a plethora of physiological and pathophysiological processes, such as Alzheimer's disease, atherosclerosis and cancer. In its dominant form it is expressed as a secretory protein (secreted CLU, sCLU). During its maturation, the sCLU-precursor is N-glycosylated and cleaved into an α- and a β-chain, which are connected by five symmetrical disulfide bonds. Recently, it has been demonstrated that besides the predominant sCLU, rare intracellular CLU forms are expressed in stressed cells. Since these forms do not enter or complete the secretory pathway, they are not proteolytically modified and show either no or only core glycosylation. Due to their sparsity, these intracellular forms are functionally poorly characterized. To evaluate the function(s) of these stress-related intracellular forms, we investigate for the first time the impact of proteolytic cleavage, differential glycosylation and the influence of the redox environment on the chaperone activity of CLU. Methods: Non-cleavable sCLU was generated by expression from a mutant construct of sCLU, in which the furin-like proprotein convertase (PC) recognition site was modified. After purification of recombinant uncleaved sCLU from the medium of over-expressing cells, we performed chaperone activity assays to compare the activities of wild-type (cleaved) and uncleaved mutant sCLU. Additionally, this approach enabled us to investigate the role of carbohydrates, the proteolytic maturation and reducing conditions on CLU chaperone activity. Further, we characterized the differentially treated CLU forms by using MALDI-TOF, CD-spectroscopy and Western blotting in addition to the functional assay. Results: We show that the PC-cleavage is dispensable for sCLU chaperone activity. Moreover, our data demonstrate that while fully deglycosylated sCLU lacks chaperone activity, partially deglycosylated sCLU is still capable of solubilizing target proteins. Most importantly, we here demonstrate for the first time that uncleaved sCLU is highly sensitive towards reducing conditions. Conclusions: Our study provides evidence that unglycosylated intracellular CLU forms cannot exhibit a chaperone activity compared to sCLU. Additionally, we support recent postulates that glycosylated intracellular CLU forms may act as a redox sensor under oxidative stress conditions. Furthermore, we conclude that the proteolytic cleavage of sCLU is important to maintain full chaperone activity, i.e. in the presence of necrosis.

Clusterin: full-length protein and one of its chains show opposing effects on cellular lipid accumulation

“…It was shown by Rohne et al . that hypoglycosylated clusterin, having just the core glycosylation, had significant chaperone-like activity and even completely deglycosylated clusterin showed minimal chaperone activity37. However, in our experiments we observe that both α-Clu and β-Clu co-aggregate with insulin when subjected to DTT-induced aggregation.…”

“…Both α-Clu and β-Clu show appreciable α-helical content (39 and 36% respectively). However, compared to the α-helical content of full-length, secretory clusterin (~62% α-helix, 7% β-sheet, ~12% turns and 20% random coil), which consists of both the chains linked by 5 disulphides, their α-helical content is significantly less37. This result suggests that long range interactions stabilize the α-helicity of some of the regions in the full length clusterin.…”

“…have reported that deglycosylation of clusterin does not significantly affect the chaperone activity of the protein40, another study by Rohne et al . showed that complete deglycosylation of clusterin, in the presence of DTT, causes up to 90% loss in the chaperone activity of the protein37. Intriguingly, clusterin is reported to have two levels of glycosylation; core glycosylation involving mannose moieties and further glycosylation of these residues with higher oligosaccharides4243.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

“…It will remove most N ‐glycans, with the exception of those containing a 3‐linked fucose residue on the reducing‐terminal GlcNAc residue. However, for some glycoproteins, not all glycosylation sites are accessible to the enzyme as recently illustrated by clusterin (apolipoprotein J) (Rohne et al, ). Reaction with PNGase F, even at relatively high concentration, only partially deglycosylated the protein.…”

Apolipoprotein J is a hepatokine regulating muscle glucose metabolism and insulin sensitivity

“…ApoJ exists as multiple protein isoforms including the 75-80 kDa highly glycosylated secreted form, and the smaller intracellular forms that are not well characterized 15,16 . The secretory isoform of ApoJ is thought to have molecular chaperone activity depending on the degree of glycosylation 17,18 and binds to specific cell surface receptors in mediating its biological effects, such as endocytosis 19,20 . Although the exact role of ApoJ in many conditions remains unclear 15 , ApoJ has been implicated in altered pathophysiologic disorders, including atherosclerosis 21 , obesity 22 , diabetes 23 , and Alzheimer's disease 24 .…”