Biochimica et Biophysica Acta. 1089 (1991) 13-20 ~ 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 0167478191001202 13 BBAEXP 92232 Molecular cloning of mouse hepatic triacylglycerol lipase: gene expression in combined lipase-deficient (cld/cld) mice Kazuhiro Oka 1 Tamotsu NakancJ 1, George T. Tkalcevic ~, Robert O. Scow 2 and W. Virgil Brown / The Laboratory of Molecular Genetics, Medlantic Research Foundation, Washington DC (U.S.A.) and 2 The Endocrinololo" Section. Laboratory of Cellular and Developmental Biolog)', National Institute of Diabetes and Digestit,e and Kidney Diseases, National Institutes of Health, Bethesda, MD (U.S.A.) (Received 24 July 1990) (Revised manuscript received 3 December 1990) Key words: Hepatic triacylglycerol lipase; eDNA sequence: Combined lipase deficiency; (Mouse) cDNA clones coding for mouse hepatic triacylglycerol lipase (HL) were isolated from a mouse liver cDNA library with a human HL eDNA as a probe° The cloned HL cDNA of 1652 nucleotides predicts a mature protein of 4188 amino acids preceded by a signal peptide of 22 amino acids. Two potential sites for N-glycosylation are identified, which are both conserved in rat and human HI,. Combined lipase deficiency (c/d) is a recessive mutation in mice, which causes the functional deficiency of HL and lipoprotein lipase, the isolated cDNA was used to study the expression of HI, gene in cld/cM mice. Northern blot analysis of total cellular RNA from livers of :id/cid and normal mice showed that there are two mRNA species for HL with the sizes of 1.8 and 1.9 kilobases in both groups. However, the mRNA for HL was more abundant in cld/cld than in normal mk'e. RNase A protection assay of HL mRNA suggested that the multiple mRNA species for IlL in dd/cld and normal mice are generated by differential utilization of polyadenylation signals and that there is no mutation in the structural gene for HL in cld/cld mice. The present study supports our hypothesis that the defect of HL activity in cld/cld mice is caused by abnormal post translational modification or processing of the Iipase. Introduction Hepatic triacylglycerol lipas¢ (HL) is a lipolytic en- zyme synthesized by bepatocytes and localized in or near hepatic sinusoids [1,2]. The exact function of this enzyme is unclear. However, studies of intravenous in- jection of antibody against HL [3-5] have suggested that this enzyme is critical for conversion of inter- mediate density lipopro~ein to low density lipoprotein. It also appears important in the hydrolysis of triacyl- glycerols in high density Iipoprotein. Since lipoprotein lipase (LPL) and HL are major enzymes involved in Abbreviations: HL, hepatic triacylglycerol lipase: LPL, lipoprotein lipase; cld, combined lipase deficiency; apo, apolipoprotein. The nuclcotide sequence reported in this paper has been submitted to the EMBL/Genbank Data Libraries under the accession number X!6735, Correspondence: K. Oka, Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza. Houston. TX 77030. U.S.A. removal of triacylglycerols of circulating lipoproteins, the defect of either or both enzymes causes abnormal lipoprotein patterns. Combined lipase deficiency (cid) is a recessive gene mutation within T/t complex of chromosome 17 in mice, which is characterized by severe functional deficiency of both LPL and HL [6-8]. Animals with the homozygous form of this mutation have normal blood lipids in utero, but develop severe hyperchylomicronemia when allowed to suckle and die within 3 days after birth. The structural gene for LPL has been mapped to mouse chromosome 8 and for HL to 9 or 11 [9]. Although the possibility of a point mutation has not been excluded, a study of the expres- sion of the LPL gene in tissues of cld/cld mice has suggested that there may be no mutation in the struct- ural gene [10]. Because the deficiency appears to be limited to these giycoproteins, there must be a common processing of the two enzymes which is linked to one or more common structural feature~ Thus, a comparison of the primary structures of HL and LPL may give clues to the defect in cld/cld mice. LPL cDNAs have been