Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21)

Abstract

Lipoprotein lipase (LPL) is a non-covalent, homodimeric, N-glycosylated enzyme important for metabolism of blood lipids. LPL is regulated by yet unknown post-translational events affecting the levels of active dimers. On co-expression of LPL with human molecular chaperones, we found that calreticulin had the most pronounced effects on LPL activity, but calnexin was also effective. Calreticulin caused a 9-fold increase in active LPL, amounting to about 50% of the expressed LPL protein. The total expression of LPL protein was increased less than 20%, and the secretion rates for active and inactive LPL were not significantly changed by the chaperone. Thus, the main effect was an increased specific activity of LPL both in cells and media. Chromatography on heparin-Sepharose and sucrose density gradient centrifugation demonstrated that most of the inactive LPL was monomeric and that calreticulin promoted formation of active dimers. Higher oligomers of inactive LPL were present in cell extracts, but only monomers and dimers were secreted to the medium. Interaction between LPL and calreticulin was demonstrated, and the effect of the chaperone was prevented by castanospermine, an inhibitor of N-glycan glucose trimming. Our data indicate an important role of endoplasmic reticulum-based chaperones for the folding/ dimerization of LPL. Lipoprotein lipase (LPL)1 hydrolyzes triglycerides in the circulating plasma lipoproteins, such as chylomicrons and very low density lipoproteins (1-3). The enzyme is produced and secreted mainly by adipocytes and muscle cells, but it is also produced in other cells such as macrophages and certain neurons. LPL is transported by yet unclear mechanisms to the vascular endothelium, where it is attached to cell surfaces through interaction with heparan sulfate proteoglycans (HSPG). The products of lipolysis are taken up for storage or oxidation in adjacent tissues. In addition, LPL can mediate internalization of intact lipoproteins by bridging them to cell surface receptors like the LDL receptor-like protein or HSPG.LPL, along with pancreatic lipase, hepatic lipase (HL), and endothelial lipase, belongs to the gene family of mammalian triglyceride lipases (2, 4). The three-dimensional structure of LPL has not yet been determined. Based on homology with pancreatic lipase, it has been proposed that LPL has two structurally distinct regions, a larger N-terminal domain containing the active site and a smaller C-terminal domain with other important functions, such as binding to lipids, LDL receptorlike protein, and HSPG (5, 6).Catalytically active LPL is a non-covalent, homodimeric glycoprotein (7, 8). In the rough endoplasmic reticulum (ER), human LPL is co-translationally glycosylated at amino acid residues Asn 43 and Asn 359 . Studies using site-directed mutagenesis and inhibitors of transport between ER and Golgi have shown that core glycosylation at residue Asn 43 is necessary for activation and release of LPL from the ER, but glycosylation at Asn 359 is not necessary (9 -11). A number of studies...

Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum

“…Additionally, since mitochondria are of pivotal importance to direct entering Ca 2+ towards the ER for its refilling [15], the contribution of such mitochondrial Ca 2+ transfer to Ca 2+ -dependent protein maturation in the ER needs to be elucidated. Therefore the efficiency of Ca 2+ -dependent protein maturation in the ER was determined utilizing LPL, the activity and secretion of which strictly depends on proper folding by Ca 2+ -sensitive chaperones and folding catalysts in the ER [11,13,21]. Consequently, LPL expression was correlated with enzyme activity as a measure of correctly folded LPL.…”

“…protein disulphide-isomerase and ERp57, inactive [9]. However, these principles of Ca 2+ -dependent protein folding have been assessed mainly on recombinant proteins [7,8,10] or by artificially applying excessive ER stress [11][12][13], while a detailed analysis of its kinetics and correlation with physiological Ca 2+ fluctuations are lacking. While there are studies that address this issue [14], it still remains questionable whether the ER protein-folding machinery is influenced by spatial and temporal dynamics of [Ca 2+ ] ER in intact cells and, if so, how efficient protein folding is maintained under physiological cell stimulation.…”

“…LPL, the major lipolytic enzyme for the hydrolysis of circulating lipoproteins, is a secretory N-linked glycoprotein, which contains two conserved disulphide bridges, and interacts with the Ca 2+ -dependent proteinfolding machinery of the ER during its maturation [11,13,21]. As only the correctly folded protein is active and secreted [11,21], LPL activity is dependent on proper maturation in the ER and it was therefore assumed to be a suitable sensor for ER chaperonedependent folding processes.…”

Mechanisms of lipase maturation

“…In particular, HL associates with the lectin chaperone CNX, as would be expected from the maturation defects encountered when glucosidase inhibitors prevent binding of HL and LPL to CNX (discussed earlier); other studies using co-immunoprecipitation have also detected HL-CNX association [75,90]. Moreover, the maturation efficiency of transfected human LPL in insect cells is greatly increased by cotransfection with mammalian CNX/CRT [91]. Along with CNX, components of the CNX/ CRT system were also identified as being associated with HL during its maturation [76], including UGGT, GII and ERp57 (Table 1).…”

Design of signal peptide bombyxin and its effect on secretory expression efficiency and levels of Helicobacter pylori urease subunit B in silkworm cells and larvae

“…The secretory expression of recombinant proteins in host cells is also a highly effective strategy. In this process, the signal peptide and certain molecular chaperones play critical roles in enhancing the secretion of recombinant proteins (Chang et al 1997;Tate et al 1999;Higgins et al 2003;Zhang et al 2003;Kato et al 2004). Although several studies have reported the successful secretion of recombinant proteins in insect cells (Chen et al 2000;Alam et al 2002), there are few reports to date on the secretion of exogenous proteins into the hemolymph of silkworm pupae or larvae.…”