Abstract
For three decades, the LPL-specific monoclonal antibody 5D2 has been used to investigate LPL structure/function and intravascular lipolysis. 5D2 has been used to measure LPL levels, block the triglyceride hydrolase activity of LPL, and prevent the propensity of concentrated LPL preparations to form homodimers. Two early studies on the location of the 5D2 epitope reached conflicting conclusions, but the more convincing report suggested that 5D2 binds to a tryptophan (Trp)-rich loop in the carboxyl terminus of LPL. The same loop had been implicated in lipoprotein binding. Using surface plasmon resonance, we showed that 5D2 binds with high affinity to a synthetic LPL peptide containing the Trp-rich loop of human (but not mouse) LPL. We also showed, by both fluorescence and UV resonance Raman spectroscopy, that the Trp-rich loop binds lipids. Finally, we used X-ray crystallography to solve the structure of the Trp-rich peptide bound to a 5D2 Fab fragment. The Trp-rich peptide contains a short α-helix, with two Trps projecting into the antigen recognition site. A proline substitution in the α-helix, found in mouse LPL, is expected to interfere with several hydrogen bonds, explaining why 5D2 cannot bind to mouse LPL.
Highlights
The LPL–specific mouse monoclonal antibody 5D2, created by the laboratory of John Brunzell [1,2,3], has been a key reagent for investigating LPL for more than 30 years. 5D2 was generated by immunizing mice with bovine LPL but binds LPL from multiple vertebrate species [2, 3]. 5D2 was initially used to develop immunoassays for LPL in human plasma [1]
Soon after Lookene et al [6] showed that LPL residues W420 and W421 were important for 5D2 binding, Chang et al [2] found, using competitive immunoassays, that 5D2 bound to a synthetic peptide corresponding to LPL’s Trprich motif but not to the sequences proposed initially by Brunzell’s group. 5D2 did not bind to a synthetic peptide corresponding to the Trp-rich motif of mouse LPL, which contained a Ser-toPro substitution at residue 418
Kristensen et al [9] demonstrated, using synthetic peptides and surface plasmon resonance (SPR) studies, that 5D2 binds to LPL’s Trp-rich motif, and they went on to quantify the impact of each amino acid residue in the Trp-rich loop for 5D2 binding affinity
Summary
The LPL–specific mouse monoclonal antibody 5D2, created by the laboratory of John Brunzell [1,2,3], has been a key reagent for investigating LPL for more than 30 years. 5D2 was generated by immunizing mice with bovine LPL but binds LPL from multiple vertebrate species (including rat LPL but not mouse LPL) [2, 3]. 5D2 was initially used to develop immunoassays for LPL in human plasma [1]. The laboratory of John Brunzell proposed, based on experiments with a LPL synthetic peptide, that 5D2 binds to LPL residues 423–432 [5], but subsequent studies cast doubt on that finding. Soon after Lookene et al [6] showed that LPL residues W420 and W421 were important for 5D2 binding, Chang et al [2] found, using competitive immunoassays, that 5D2 bound to a synthetic peptide corresponding to LPL’s Trprich motif (residues 411–423) but not to the sequences proposed initially by Brunzell’s group (residues 423–432). Additional studies with antibody 5D2 led them to infer that LPL’s catalytic activity depends on the assembly of LPL into homodimers [3]. Using 5D2based ELISAs and density gradient ultracentrifugation studies, Beigneux et al [18] showed that freshly secreted catalytically active LPL is monomeric. The fact that freshly secreted LPL, as well as low concentrations of purified LPL, are monomeric was confirmed by highly standardized density gradient ultracentrifugation studies [18]
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