Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity

Abstract

This article is available online at http://www.jlr.org (1). Together, these actions make LPL a vital enzyme for the clearance of triglycerides from circulation (2, 3). LPL deficiency (LPLD) is a rare but serious condition caused by homozygous or combined heterozygous loss-of-function mutations in LPL (4). Individuals with LPLD suffer from pronounced accumulation of triglyceride-rich lipoproteins in the plasma, which puts affected individuals at high risk for recurrent, acute pancreatitis (5). Apart from strictly limiting dietary fat, the only treatment option was a gene therapy product known as Glybera, which consists of a gain-of-function LPL variant (LPL S447X) delivered via adeno-associated virus vectors (6). However, at a cost of close to $1 million per patient, Glybera proved too expensive and was withdrawn from the market (7). Volanesorsen, an antisense oligonucleotide drug targeting ApoCIII, decreased triglycerides in individuals with familial chylomicronemia syndrome, but is not currently Food and Drug Administration (FDA)-approved (8). Additional treatment options for LPL-related diseases are therefore needed. Protein therapeutics are a key class of medicines utilized to treat multiple diseases. FDA-approved therapeutics include monoclonal antibodies, replacement enzymes, coagulation factors, hormones, and other plasma proteins. A classic example of this technology is the use of purified insulin for the treatment of both T1D and T2D, which has been in use for nearly 100 years (9). There are also a number of metabolic disorders that have enzyme-replacement therapy (ERT) treatments such as Gaucher's disease, Fabry disease, and Wolman disease (10). Although there are Abstract LPL is a secreted enzyme that hydrolyzes triglycerides from circulating lipoproteins. Individuals lacking LPL suffer from severe hypertriglyceridemia, a risk factor for acute pancreatitis. One potential treatment is to administer recombinant LPL as a protein therapeutic. However, use of LPL as a protein therapeutic is limited because it is an unstable enzyme that is difficult to produce in large quantities. Furthermore, these considerations also limit structural and biochemical studies that are needed for large-scale drug discovery efforts. We demonstrate that the yield of purified LPL can be dramatically enhanced by coexpressing its maturation factor, LMF1, and by introducing novel mutations into the LPL sequence to render it resistant to proteolytic cleavage by furin. One of these mutations introduces a motif for addition of an N-linked glycan to the furin-recognition site. Furin-resistant LPL has previously been reported, but is not commonly used. We show that our modifications do not adversely alter LPL's enzymatic activity, stability, or in vivo function. Together, these data show that furin-resistant LPL is a useful reagent for both biochemical and biomedical studies.

A lipoprotein lipase–GPI-anchored high-density lipoprotein–binding protein 1 fusion lowers triglycerides in mice: Implications for managing familial chylomicronemia syndrome

“…Coexpression of chaperones is one of the approaches used to optimize the expression of recombinant proteins (14). Cotransfection with the LPL chaperone protein, LMF1, has been reported to improve the yield of recombinant LPL (15). We, on the contrary, observed that cotransfection with LMF1 did not substantially improve LPL yield, and the purified protein was still highly aggregated ( Fig.…”

Mapping the sites of the lipoprotein lipase (LPL)–angiopoietin-like protein 4 (ANGPTL4) interaction provides mechanistic insight into LPL inhibition

“…LPL is inactivated by furin protease cleavage into the N-and C-terminal domains at the consensus sequence "RAKR" (residues 294 -297). Although we were unable to generate enzymatically active chimeric mutants at this site, we have shown that furin-resistant LPL point mutants, R297N and R297N/S298C, are sensitive to nANGPTL4 inhibition (50). These data suggest residues Arg-297 and Ser-298 may not be necessary for nANGPTL4-mediated inhibition of LPL.…”

“…S3A). Furin protease recognizes the consensus sequence "RXX(K/ R)" present in LPL at residues 294 -297, resulting in inactive Nand C-terminal cleavage fragments (49,50). In HL, the furin consensus sequence is disrupted by a proline residue and protected from proteolytic cleavage in this region (residues 314 -334).…”

Comparison of angiopoietin-like protein 3 and 4 reveals structural and mechanistic similarities

“…Bovine LPL (UniProt accession ID P11151) was purified from raw cow’s milk using heparin chromatography as reported ( 39 ). Furin-resistant human LPL (UniProt accession ID P06858) was purified as reported ( 40 ).…”

“…ANGPTL4 was probed with a polyclonal rabbit anti-ANGPTL4 antibody (BioVendor) using a 1:5000 dilution and detected with a horseradish peroxidase-conjugated donkey anti-rabbit antibody (Southern Biotech) using a 1:5000 dilution. Western blots were developed as previously described (40). Conflict of interest-The authors declare that they have no conflicts of interest with the contents of this article.…”