Hepatic lipase: a member of a family of structurally related lipases.

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.

Regulation of lipoprotein lipase immunological study of adipose tissue.

An antibody to purified rat heart lipoprotein lipase was used to determine the relative specific activities of adipose tissue lipoprotein lipase from fed and fasted rats. The antibody was immobilized by coupling it to a Sepharose gel. This antibody bound approx. 80% of the lipoprotein lipase activity of extracts of rat adipose tissue. When the extracts were separated by gel chromatography into two lipase activity fractions (lipoprotein lipase "a" and lipoprotein lipase "b") and these fractions incubated with the antibody, only 10% of the lipoprotein lipase "a" activity was bound by the highest antibody concentration employed, whereas 93% of the lipoprotein lipase "b" was bound by the same amount of antibody. Increasing amounts of antibody incubated with extracts of adipose tissue of fed or fasted rats yielded similar titration curves. When a constant amount of antibody was incubated with increasing amounts of the adipose extracts, no significant difference was noted between extracts from fed and fasted animals. The data indicate that the high lipoprotein lipase activity of adipose tissue of fed rats, compared with that of rats fasted overnight, results from the presence of more lipoprotein lipase protein.

Metabolic studies of adipose tissue in acute uremia.

The effect of acute uremia on lipoprotein lipase (LPL) activity in rat adipose tissue and on the response of the isolated adipocytes to insulin was assessed. LPL activity in adipose tissue and in adipocytes of the uremic rats was decreased compared with values in sham-operated controls. Also, the adipocytes from uremic rats released significantly less than control amounts of LPL. In contrast, glucose oxidation by adipocytes isolated from uremic rats was not different from controls, and there was no difference in insulin binding or in insulin-stimulated glucose oxidation in the two groups. Triglyceride injected into the uremic rats was cleared at about half the control rate. Thus, the specific reduction in LPL activity in adipose tissue may be responsible, at least in part, for the defective removal of triglyceride. However, it is unlikely that the reduced LPL is due to a generalized toxic effect of uremia on adipose tissue since no significant alteration in insulin binding and glucose oxidation was found.

Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.

Hepatic lipase and lipoprotein lipase hydrolyze fatty acids from triacylglycerols and are critical in the metabolism of circulating lipoproteins. The two lipases are similar in size and amino acid sequence but are distinguished by functional differences in substrate preference and cofactor requirement. Presumably, these distinctions result from structural differences in functional domains. To begin localization of these domains, a chimeric lipase was constructed composed of the N-terminal 329 residues of rat hepatic lipase linked to the C-terminal 136 residues of human lipoprotein lipase. The chimera hydrolyzed both monodisperse short-chain (esterase) and emulsified long-chain (lipase) triacylglycerol substrates with catalytic and kinetic properties closely resembling those of native hepatic lipase. However, monoclonal antibodies to lipoprotein lipase inhibited the lipase activity, but not the esterase function, of the chimera. Therefore, the chimeric molecule is a functional lipase and contains elements and characteristics from both parental enzymes. It is proposed that the N-terminal domain, containing the active center from hepatic lipase, governs the catalytic character of the chimera, and the C-terminal domain is essential for hydrolysis of long-chain substrates.

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.

Evidence for independent genetic regulation of heart and adipose lipoprotein lipase activity.

The relationship between the genes controlling heart and adipose lipoprotein lipase in fasted animals has been studied. 32 inbred mouse strains were tested for variations in heart or adipose specific activity and thermolability. The survey revealed that specific activity of heart and adipose lipoprotein lipase varied as much as 3-fold and 20-fold, respectively. In thermolability, up to a 2-fold variation was observed in the lipase in each tissue. The correlation coefficient between variations in heart and adipose lipase was apparently not significant for both parameters studied. Additional studies were performed in two strains, BALB/c and C57BL/6, along with the recombinant inbred set derived from them. The two strains did not show genetic variation for lipoprotein lipase thermolability, although the inactivation rate of heart lipase was higher than that of adipose lipase. However, BALB/c and C57BL/6 displayed significant differences in their levels of lipoprotein lipase specific activity. Thus, strain C57BL/6 showed higher heart activity when compared to BALB/c, whereas the latter showed higher adipose lipase activity when compared to C57BL/6, i.e. an inverse relationship. The specific activity levels of heart and adipose lipoprotein lipase in the recombinant inbred strains derived from BALB/c and C57BL/6 exhibited independent inheritance. Thus, in adipose tissue, a single major gene seems to control the variation observed, while the inheritance pattern of heart activity could imply involvement of more than one gene. Moreover, two out of the seven recombinant strains showed distinct recombinant phenotypes, indicating that separate unlinked genes control the variations found in heart and adipose activity. We conclude that the expression of heart and adipose lipoprotein lipase activity is under independent genetic control.

A molecular biology-based approach to resolve the subunit orientation of lipoprotein lipase.

The subunit orientation of a dimeric enzyme influences the mechanism of action and function. To determine the subunit arrangement of lipoprotein lipase (LPL), a molecular biology-based approach was initiated. An eight amino acid linker region was engineered between two LPL monomers and expressed in COS-7 cells. The resultant tandem-repeat molecule (LPLTR) was lipolytically active and had kinetic parameters, salt inhibition, cofactor-dependent activity, heparin-binding characteristics, and a functional unit size very similar to the expressed native human enzyme. By these criteria, LPLTR was the functional equivalent of native LPL. Considering the length of the linker peptide (no more than 24 A), monomers in the tethered molecule were restricted to a head-to-tail subunit arrangement. Since LPLTR demonstrated native enzyme-like properties while constrained to this subunit arrangement, these results provide the first compelling evidence that native LPL monomers are arranged in a head-to-tail subunit orientation within the active dimer. Thus, LPL function in physiology, lipolysis, and binding to cell-surface components must now be addressed with this subunit orientation in mind. The utility of the tandem-repeat approach to resolve the subunit arrangement of an obligate dimer has been demonstrated with LPL and could be generalized for use with other oligomeric enzymes.

Lipoprotein lipase domain function.

Human lipoprotein lipase (LPL) monomer consists of two domains, a larger NH2-terminal domain that contains catalytic residues and a smaller COOH-terminal domain that modulates substrate specificity and is a major determinant of heparin binding. Analyses of NH2-terminal domain function were performed after site-directed mutagenesis of the putative active-site serine residue, while COOH-terminal domain function was assessed following reaction with a monoclonal antibody. The native enzyme and mutant LPL in which serine 132 was replaced with alanine, cysteine, or glycine were transiently expressed in COS-7 cells. Mutant proteins were synthesized and secreted at levels comparable to native LPL; however, none of the mutants retained enzymatic activity. The mutant with alanine replacing serine 132 was purified and shown to be inactive with both esterase and lipase substrates; however, binding to a 1,2-didodecanoyl-sn-glycero-3-phosphatidylcholine monolayer was comparable to native LPL. These results are consistent with a catalytic, and not a lipid binding, role for serine 132. To investigate the function of the smaller COOH-terminal domain, LPL lipolytic and esterolytic activities as well as heparin binding properties were determined after reaction with a monoclonal antibody specific for this domain. Lipolytic activity was inhibited by the monoclonal antibody, whereas esterolytic activity was only marginally affected, indicating that the LPL COOH-terminal domain is required for lipolysis, perhaps by promoting interaction with insoluble substrates. Also, the affinity of antibody-reacted LPL for heparin was not significantly different from that of LPL alone, suggesting that (i) the heparin-binding site is physically distinct from the COOH-terminal domain region required for lipolysis and (ii) binding of antibody did not cause dimer dissociation. A model is proposed for the two LPL domains fulfilling different roles in the lipolytic process.

Lipoprotein lipase: size of the functional unit determined by radiation inactivation.

Radiation inactivation was used to determine the functional molecular weight of lipoprotein lipase (LPL) in rat heart and adipose tissues. This technique reveals the size of the smallest unit required to carry out the enzyme function. Supernatant fractions of the tissue homogenates were exposed to high energy electrons at -135 degrees C. LPL activity showed a simple exponential decay in all samples tested. Because changes in nutritional state shift the distribution of LPL between the capillary endothelial and parenchymal cells within heart and adipose tissues, fasted and refed rats were used for the radiation studies. The functional molecular weight was calculated to be 127,000 +/- 15,000 (mean +/- SD) daltons for heart and adipose. Thus, the smallest unit required for enzyme function was the same in both of these tissues and did not vary with nutritional state. The data suggest that, compared with LPL monomer sizes reported in the range 55,000 to 72,000, this active unit constitutes a dimer.

Subdomain chimeras of hepatic lipase and lipoprotein lipase. Localization of heparin and cofactor binding.

To specify and localize carboxyl-terminal domain functions of human hepatic lipase (HL) and human lipoprotein lipase (LPL), two subdomain chimeras were created in which portions of the carboxyl-terminal domain were exchanged between the two lipases. The first chimera (HL-LPLC1) was composed of residues 1-344 of human HL, residues 331-388 of human LPL, and residues 415-476 of human HL. The second chimera (HL-LPLC2) consisted of just two segments, residues 1-414 of human HL and residues 389-448 of human LPL. These chimeric constructs effectively divided the HL C-terminal domain into halves, with corresponding LPL sequences either in the first or second portion of that domain. Both chimeras were lipolytically active and hydrolyzed triolein emulsions to a similar extent compared with native HL and LPL. Heparin-Sepharose chromatography demonstrated that HL-LPLC1 and HL-LPLC2 eluted at 0.80 and 1.3 M NaCl, respectively, elution positions that corresponded to native HL and LPL. Hence, substitution of LPL sequences into the HL carboxyl-terminal domain resulted in the production of functional lipases, but with distinct heparin binding properties. In addition, HL-LPLC2 trioleinase activity was responsive to apoC-II activation, although the -fold stimulation was less than that observed with native LPL. Moreover, an apoC-II fragment (residues 44-79) was specifically cross-linked to LPL and HL-LPLC2, but not to HL or HL-LPLC1. Finally, both chimeras hydrolyzed phospholipid with a specific activity similar to that of HL, which was unaffected by the presence of apoC-II. These findings indicated that in addition to a region found within the amino-terminal domain of LPL, apoC-II also interacted with the last half of the carboxyl-terminal domain (residues 389-448) to achieve maximal lipolytic activation. In addition, the relative heparin affinity of HL and LPL was determined by the final 60 carboxyl-terminal residues of each enzyme.