David L. Ebert

The regulation of hepatic lipase and cholesteryl ester transfer protein activity in the cholesterol fed rabbit

Hepatic lipase (HL) and cholesteryl ester transfer protein (CETP) activities are both increased in the rabbit by cholesterol feeding. The in vivo regulation of HL and CETP were explored by examining changes in specific steady-state mRNA levels upon cholesterol feeding. On feeding rabbits cholesterol, HL activity increased 3-fold after 2 days and remained at 2.6-times the control value at 28 days. Specific rabbit HL mRNA levels were assessed by dot blot analysis of liver poly (A)+ RNA hybridized with the human HL cDNA. No significant changes in liver HL mRNA accompanied the increase in activity seen at days 2 and 7. At day 28 a modest rise of 46% was observed. A significant rise in CETP activity, evident 7 days after the commencement of cholesterol feeding, was maintained until day 28 when it was 2.4-times the control value. Using the human CETP cDNA as probe, rabbit liver CETP mRNA was also found to increase by day 7, rising to 3.7-times control by day 28. The strong temporal relationship between the rise in CETP activity and mRNA (r = 0.55, P = 0.02) suggests that the regulation of CETP may be primarily effected by the levels of specific mRNA. In contrast, the discordance between levels of lipase activity and mRNA suggests that post-transcriptional events may be more important in the regulation of HL in the cholesterol fed rabbit.

Chapter 7 Defining the Intracellular Localization Pathways followed by Lysosomal Enzymes in Dictyostelium discoideum

Publisher Summary This chapter describes the biochemical and genetic methods used to define both the intracellular pathways followed by lysosomal enzymes and the molecular nature of the “sorting” signals found on the proteins. Using biochemical methods, the lysosomal enzymes α-man and β-glu in Dictyostelium are synthesized as precursor polypeptides that are cotranslationally translocated into the lumen of the endoplasmic reticulum (ER) and N-glycosylated. The precursors move at different rates from the RER to the Golgi complex, where they are sulfated and sorted into two classes. One class, containing the majority of the precursor polypeptides, is directed to lysosomes where they are rapidly cleaved to mature forms of the enzymes. The other class of precursors is rapidly secreted from cells. The different intracellular transport rates for the α-man and β-glu precursors support the existence of a transport-mediating receptor. The genetic approach involved the isolation and characterization of mutants altered in the secretion, modification, processing, and/or localization of lysosomal enzymes. Through biochemical analysis of these mutants, one can identify and characterize the molecular components involved in the processing and transport of this group of enzymes.

Apolipoprotein B and a second major gene locus contribute to phenotypic variation of spontaneous hypercholesterolemia in pigs.

The Lpb5 apolipoprotein B (apoB) allele occurs in pigs with spontaneous hypercholesterolemia. Low-density lipoprotein (LDL) from these pigs binds to the LDL receptor with a lower affinity and is cleared from the circulation more slowly than control pig LDL. However, the severity of hypercholesterolemia in pigs with the mutant apoB allele is highly variable. This study aimed to determine the metabolic basis for the phenotypic heterogeneity among Lpb5 pigs. Lpb5 pigs were divided into two groups: those with plasma cholesterol greater than 180 mg/dL (Lpb5.1) and those with plasma cholesterol less than 180 mg/dL (Lpb5.2). LDL from both Lpb5.1 and Lpb5.2 pigs was catabolized in vivo and in vitro at a similarly reduced rate. The difference in plasma cholesterol between the two phenotypic groups was in part due to a higher buoyant LDL production rate in Lpb5.1 pigs than in Lpb5.2 pigs. The in vivo LDL receptor status was evaluated by measuring the catabolism of LDL chemically modified to abrogate LDL receptor binding. Approximately 50% of LDL clearance in normal and Lpb5.2 pigs was via the LDL receptor; in Lpb5.1 pigs, 100% of LDL clearance was LDL receptor independent. Quantitative pedigree analysis of the segregation of the plasma cholesterol phenotype suggested that two major gene loci (the apoB locus and a second apparently unlinked locus) contribute to the determination of plasma cholesterol levels in this pig population.

Biogenesis of lysosomal enzymes in the α-glucosidase II-deficient modA mutant of Dictyostelium discoideum: Retention of α-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of α-mannosidase or β-glucosidase

Abstract The endoplasmic reticulum-localized enzyme α-glucosidase II is responsible for removing the two α-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum . Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, α-mannosidase and β-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant α-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of α-mannosidase and β-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N -acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.

Alterations to N-linked oligosaccharides which affect intracellular transport rates and regulated secretion but not sorting of lysosomal acid phosphatase in Dictyostelium discoideum

The importance of N-linked oligosaccharides and their associated modifications in the transport, sorting, and secretion of lysosomal acid phosphatase was investigated using three mutant Dictyostelium cell lines. These mutants synthesize altered N-linked oligosaccharides with the following properties: (i) in strain HL244 carbohydrate side chains lack mannose 6-sulfate residues, (ii) in strain M31 the side chains retain the two alpha-1,3-linked glucose residues resulting in less sulfate and methylphosphate modifications, and (iii) in strain HL243 the nonglucosylated branches are missing three of the outer mannose sugars and the oligosaccharides contain fewer sulfate and phosphate modifications. Lysosomal enzymes in both HL243 and HL244 are also missing a shared epitope termed common antigen-1 (CA-1), which consists in part of mannose 6-sulfate moieties. No increases were observed in the secretion of radiolabeled acid phosphatase or acid phosphatase activity during growth in any of the mutant cell lines, suggesting that the enzyme was correctly sorted to lysosomes. In support of this, Percoll gradient fractionations and indirect immunofluorescence microscopy indicated that acid phosphatase was transported to lysosomes in all cell lines. However, radiolabel pulse chase protocols indicated that newly synthesized acid phosphatase was transported out of the endoplasmic reticulum (ER) and into lysosomes at a two- to threefold slower rate in HL243 and at a sixfold slower rate in M31. The rate of transport of acid phosphatase from the ER to the Golgi was reduced only twofold in M31 as determined by digestion of newly synthesized enzyme with endoglycosidose H. This suggests that certain alterations in carbohydrate structure may only slightly affect transport of the enzyme from the ER to the Golgi but these alterations may greatly delay transport from the Golgi or post-Golgi compartments to lysosomes. Finally all three mutants secreted acid phosphatase at significantly lower rates than the wild-type strain when growing cells were placed in a buffered salt solution (conditions which stimulate the secretion of mature lysosomally localized enzymes). In contrast, alpha-mannosidase was secreted with similar kinetics from the mutant and wild-type strains. Together, these results suggest that the mechanism(s) operating to sort acid phosphatase in Dictyostelium can tolerate a wide range of changes in N-linked oligosaccharides including a reduction in phosphate and the absence of CA-1 and sulfate, while in contrast, these same alterations can profoundly influence the rate of transport of acid phosphatase from the ER and post-ER compartments to lysosomes as well as the secr