Cloning of rat hepatic lipase cDNA: evidence for a lipase gene family.

Abstract

Clones for rat hepatic lipase were isolated by probing a rat liver cDNA library in Agtll with an oligonucleotide synthesized on the basis of a partial peptide sequence. The cloned messenger codes for a protein of 472 amino acids plus a hydrophobic leader sequence of 22 amino acids. The unglycosylated protein has a predicted molecular weight of 53,222 and contains two potential sites for N-glycosylation. The protein bears striking regions of homology with other known lipases and contains peptide sequences that have been implicated in lipid binding. The homologous mRNA is present in liver tissue but no detectable mRNA is observed in the adrenal gland, despite the reported presence of hepatic lipase in both the liver and the adrenal gland. No mRNA was seen in any of a variety of other tissues.Hepatic lipase, localized primarily on the sinusoidal surfaces of the liver, functions in the metabolism of circulating lipoproteins (1). Hepatic lipase activity has also been detected in several extrahepatic tissues, including adrenal gland and ovary (2-4). The presumed function of the enzyme is the hydrolysis of triglycerides in intermediate density lipoproteins (IDL) and of phospholipids in high density lipoproteins (HDL2) (5). The capacity ofpurified hepatic lipase to catalyze hydrolysis of phospholipids and mono-, di-, and triglycerides (6, 7) is consistent with the putative function. The action of hepatic lipase on HDL2 is presumed to result in the production of HDL3. Also, hepatic lipase may participate in the clearance of circulating very low-density lipoproteins (8) and chylomicron remnants (9).Hepatic lipase from rat liver has been purified to homogeneity (7, 10). The enzyme has an apparent molecular weight of53,000 (7,11) cDNA Screening and Analysis. Partial amino acid sequences were determined from the digestion of purified hepatic lipase with subtilisin (11). A single 42-base oligonucleotide probe was constructed on the basis of a 14 amino acid sequence ( Fig. 1) and codon usage tables for mammalian genes (13). In addition to codon usage assumption, G-T base pairing was allowed in designing the probe. A total of 9 x 105 plaques were screened with the end-labeled 42-base oligonucleotide probe. Plaque replicas on nitrocellulose filters were made by the method of Benton and Davis (14). The filters were baked at 80'C for 2 hr and incubated for 3 hr at 370C in 30% (vol/vol) formamide/900 mM NaCl/90 mM trisodium citrate/5 x concentrated Denhardt's solution/50 mM sodium phosphate, pH 6.5, containing salmon sperm DNA at 100 pkg/ml, in a total volume of 200 ml. The 32P-labeled probe (2 x 108 cpm/Ag) was hybridized to the filters for 17 hr at 370C in 200 ml of the same solution. The filters were washed twice at room temperature in 500 ml of 150 mM NaCl/15 mM trisodium citrate/0.1% NaDodSO4 for 30 min per wash, and then six times at 370C in 300 ml of the same solution for 30 min per wash. The filters were exposed to Du Pont Cronex 4 film with Du Pont Hi-Plus intensifying screens at -70'C for 20 hr. DNA from clones t...

Characterization of cDNA encoding the mouse hepatic triglyceride lipase and expression by in vitro translation

“…the mHL sequence from amino acid positions I to 22 refers to a putative hydrophobic leader sequence. Therefore the amino terminus of mature mHL begins probably at the glycine residue after the possible leader sequence as identified by protein sequencing of rHL [7]. The calculated isoelectric point of maturc mHL is 8.14 and the estimated molecular weight is 54 958 which is similar to that determined experimentally by in vitro translation in presence of membranes (Fig.…”

“…These data indicate that (i) all three enzymes are similar in protein structure, (ii) mHL is more closely related to rHL, and (iii) that mHL is probably also similar in genomic organization to hHL. Similar to the signal sequence described for rHL [7]. hHL [8] and porcine pancreatic lipase [13].…”

“…In addition, highly conserved are the sequences of the presumptive interfacial lipid binding site (amino acids 142-156) (Fig. 2) acids 169-233) [7]. The motif Gly-Xaa-Ser-Xaa-Gly which is localized within the interfacial lipid binding site of mHL is also present in rHL and hHL.…”

“…h(m,r)HL, human (mouse, rat) hepatic triglyceride lipase and rHL are part of a multigene family [7,8]. Here we report on the cloning and sequence determination of the mouse hepatic triglyceride lipase (mHL), on the comparison of the derived amino acid sequence with the sequence of rat specific and human specific hepatic triglyceride lipase.…”

Hepatic lipase gene expression is transiently induced by gonadotropic hormones in rat ovaries

“…Random-primed cDNA was amplified for 30 -35 cycles using different HL-specific primers. HL-1 (5%-GTG GGC ATC AAA CAG CCC-3%, nt 712-729; numbering according to the rat cDNA sequence [15] and HL-2 (5%-CAG ACA TTG GCC CAC ACT-3%, nt 1307-1289) were used in quantitative PCR. The sense oligonucleotides HL-12 (5%-TGG CTT GCT AGA AAC CTG G-3%, nt 297-315), HL-13 (5%-TGT CAT GAT CAT CCA CGG G-3%, nt 267-285) and INT (5%-GCA TTG TCC TTG AGC CTG AG-3, nt-112 to − 93 according to the sequence upstream of exon 3 in adrenal HL mRNA [12]) were used in combination with the antisense oligonucleotide HL-9 (5%-GGC ATC ATC TGG AGA AAG GC-3%, nt 660-641) to determine the 5%-end of the HL mRNA.…”