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Domain Exchange: Characterization of a Chimeric Lipase of Hepatic Lipase and Lipoprotein Lipase

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Domain Exchange: Characterization of a Chimeric Lipase of Hepatic Lipase and Lipoprotein Lipase Proc. Natl. Acad. Sci. USA Vol. 88, pp. 11290-11294, December 1991 Biochemistry Domain exchange: Characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase HOWARD WONG*, RICHARD C. DAVIS, JUDITH NIKAZY, KIMBERLY E. SEEBART, AND MICHAEL C. SCHOTZ Lipid Research, Veterans Administration Wadsworth Medical Center, Los Angeles, CA 90073; and Department of Medicine, University of California, Los Angeles, CA 90024 Communicated by Daniel Steinberg, September 16, 1991 ABSTRACT Hepatic lipase and lipoprotein lipase hydro- result in inactive enzyme, and, in the absence of crystal lyze fatty acids from triacylglycerols and are critical in the structure, the precise reason for inactivation remains open to metabolism of circulating lipoproteins. The two lipases are conjecture. In many instances, global or indirect effects of similar in size and amino acid sequence but are disished these mutations are difficult to distinguish from altered by functional differences in substrate preference and cofactor chemical reactivity of critical residues or gross misfolding requirement. Presumably, these distinctions result from struc- resulting in inactivation. tural differences in functional domains. To begin localization of For these reasons, we have used an alternative strategy to these domains, a chimeric lipase was constructed composed of investigate lipase structure-function relationships: intact do- the N-terminal 329 residues of rat hepatic lipase linked to the mains were exchanged to assess functional roles played by C-terminal 136 residues of human lipoprotein lipase. The large structural components. The strategy was based on chimera hydrolyzed both monodisperse short-chain (esterase) reports describing the modular organization of proteins (14- and emulsified long-chain (lipase) triacylglycerol substrates 16), with the intent of constructing a catalytically active with catalytic and kinetic properties closely resembling those of chimeric lipase. Aside from important evolutionary consid- native hepatic lipase. However, monoclonal antibodies to lipo- erations, the modular organization of proteins provides a protein lipase inhibited the lipase activity, but not the esterase means to investigate interchangeability of functional do- function, of the chimera. Therefore, the chimeric molecule is a mains. We were motivated by the observation that members functional lipase and contains elements and characteristics of the lipase gene family displayed different functional prop- from both parental enzymes. It is proposed that the N-terminal erties, yet amino acid homology was quite high. For example, domain, containing the active center from hepatic lipase, lipoprotein lipase is a much less efficient phospholipase than governs the catalytic character of the chimera, and the C-ter- HL, even though these enzymes have >50%6 amino acid minal domain is essential for hydrolysis of long-chain sub- identity. Basic differences in lipase characteristics provide an strates. opportunity to evaluate the contribution of individual struc- tural domains to enzyme function. Hepatic lipase (HL) and lipoprotein lipase are enzymes Here we describe the construction and characteristics of a critical to processing of circulating lipoproteins. Together chimeric lipase composed of the rat HL catalytic domain with pancreatic lipase, they form a highly homologous, but (residues 1-329) directly linked to the human lipoprotein dispersed, gene family (1-3). Vitellogenin, a yolk protein, lipase C-terminal domain (residues 313-448). The junction also shares substantial homology with these lipases but lacks site for the chimera was placed within a region analogous to enzymatic function (4, 5). The three-dimensional structure of that which joins the two domains of human pancreatic lipase pancreatic lipase, derived from x-ray diffraction patterns (6), (6) and corresponds in the cDNA to the splice site between shows two independent structural domains joined by a short exons 6 and 7 of both lipoprotein lipase (3) and HL (17). The spanning region. A similar three-dimensional structure is chimera displayed catalytic properties of native HL with inferred for the other gene family members, HL and lipopro- regard to both lipase substrates (emulsified long-chain triac- tein lipase, because of the high amino acid sequence homol- ylglycerols) and esterase substrates (monodisperse short- ogy (2), similarity in the location and number of disulfide chain triacylglycerols). However, monoclonal antibody to bonds (7), and comparable function with regard to neutral lipoprotein lipase inhibited chimera lipase activity without lipid substrates. The putative pancreatic lipase catalytic affecting esterase function. These results indicated that the center, formed by a triad of serine, histidine, and aspartate, chimera was a functional lipase and possessed characteristics is located in the globular N-terminal domain. The smaller from both parental enzymes. C-terminal domain, although assigned no function, is super- ficially similar in both size and structure to fatty acid-binding protein (FABP) (8). MATERIALS AND METHODS The effect of mutations on the function of HL (9, 10) and Chimera Construction. A variation (18) of the polymerase lipoprotein lipase (11) has been reported. N-linked glycosy- chain reaction (PCR) was used to selectively amplify and link lation was required for lipoprotein lipase activity (11) but not appropriate segments ofthe cDNAs for rat HL (2) and human for HL activity (10), and replacement of residues at or near lipoprotein lipase (19). The N-terminal primer (Xba5PrHL: the putative active site resulted in the production of catalyt- 5 '-TGACTCTAGATGGGAAATCACCTCCAAATC-3') ically incompetent proteins (9, 12). In addition, naturally contained an Xba I restriction site and the first 21 coding occurring mutations, (deletions, duplications, truncations, nucleotides of the rat HL cDNA (2). The splicing primer and substitutions) have been characterized for both lipases (rHL329LPL313: 5'-CGAGCCCAGTCCCCCTTTAAAGT- (13). However, analyses ofthis kind often cannot fully define CTTCCATTACCAAGTAAAG-3') consisted of 21 nucleo- structure-function relationships. Such mutations commonly tides encoding amino acids 323-329 of rat HL (2) followed by 21 nucleotides encoding amino acids 313-319 of human The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: HL, hepatic lipase; apo, apolipoprotein. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11290 Downloaded by guest on September 27, 2021 Biochemistry: Wong et al. Proc. Natl. Acad. Sci. USA 88 (1991) 11291 lipoprotein lipase (19). The C-terminal primer (Bam3PLPL: Active-Site C-Terminal 5'-ACGTGGATCCGAATTCACATGCCGTTCTTTG-3') Domain Domain contained a BamHI restriction site followed by reverse complement for nucleotides 1623-1643 in the 3' untranslated region of human lipoprotein lipase cDNA (19). The chimeric and parental lipase cDNAs were cloned in the expression vector pSVL (Pharmacia), and the structure of the chimera was confirmed by complete nucleotide sequencing. rHL Expression. By use of Lipofectin (BRL) for transfection, LPL the lipases were transiently expressed in COS-7 cells (20) according to the normal lipofection protocol (21) except that HL-chimera during the last 18-24 hr, heparin (Sigma) was added to the tissue culture medium at 20 units/ml. The culture medium FIG. 1. Structure of the chimeric lipase. Rat HL (rHL; open bar) was concentrated in Centricon-30 microconcentrators (Am- and human lipoprotein lipase (LPL; solid bar) are shown aligned at icon) and stored at -80'C until assayed for enzymatic activ- their putative catalytic serine residues (S). HL'-chimera consists of ity. the first 329 amino acids of rat HL (2) linked to the 136 C-terminal Genomic Sequencing. Sequencing of rat HL genomic se- residues from human lipoprotein lipase (19). At the top is a lipase quences was accomplished by PCR amplification. Primers monomer model derived from the three-dimensional structure of XbaExo6FWD (5'-CGATCTAGAGCCATAACCCAGAC- pancreatic lipase, a closely related member ofthe lipase gene family. and BamExo6REV (5'-AGCTGGATCCT- The N-terminal domain contains the putative catalytic serine residue, CATCAAAT-3') while the C-terminal domain consists of eight antiparallel ,(-strands TAAAGGGGGACTGGGCTCG-3') were designed to am- present as two orthogonal sheets (6) similar in size and conformation plify rat HL sequences between codons for Ala-249 and to FABP (8). Lys-329, approximately corresponding to exon 6 of human HL (17). Amplification from Sprague-Dawley rat genomic ever, several differences with the published rat HL cDNA DNA produced a single band of appropriate size upon sequence were noted in exon 6. The same differences were electrophoresis in 1% agarose. DNA from the band was found upon resequencing the rat HL cDNA template clone. purified (22) and used in two sequencing strategies: (i) direct To determine whether these differences reflected true ge- sequencing using an internal primer spanning codons for nomic sequence, exon 6 ofHL was sequenced following PCR Leu-264 to Ser-272 in the sense direction and (ii) sequencing amplification from rat genomic DNA. All sequence differ- of plasmid DNA after cloning of the PCR product into the ences detected in the chimera (Fig. 2) were also observed in Xba I and BamHI restriction sites ofpSVL. Sequencing with genomic
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