K H Weisgraber

A role for apolipoprotein E, apolipoprotein A-I, and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve.

Recent work has demonstrated that apo E secretion and accumulation increase in the regenerating peripheral nerve. The fact that apoE, in conjunction with apoA-I and LDL receptors, participates in a well-established lipid transfer system raised the possibility that apoE is also involved in lipid transport in the injured nerve. In the present study of the crushed rat sciatic nerve, a combination of techniques was used to trace the cellular associations of apoE, apoA-I, and the LDL receptor during nerve repair and to determine the distribution of lipid at each stage. After a crush injury, as axons died and Schwann cells reabsorbed myelin, resident and monocyte-derived macrophages produced large quantities of apoE distal to the injury site. As axons regenerated in the first week, their tips contained a high concentration of LDL receptors. After axon regeneration, apoE and apoA-I began to accumulate distal to the injury site and macrophages became increasingly cholesterol-loaded. As remyelination began in the second and third weeks after injury, Schwann cells exhausted their cholesterol stores, then displayed increased LDL receptors. Depletion of macrophage cholesterol stores followed over the next several weeks. During this stage of regeneration, apoE and apoA-I were present in the extracellular matrix as components of cholesterol-rich lipoproteins. Our results demonstrate that the regenerating peripheral nerve possesses the components of a cholesterol transfer mechanism, and the sequence of events suggests that this mechanism supplies the cholesterol required for rapid membrane biogenesis during axon regeneration and remyelination.

A-IMilano apoprotein. Decreased high density lipoprotein cholesterol levels with significant lipoprotein modifications and without clinical atherosclerosis in an Italian family.

Significant hypertriglyceridemia with a very marked decrease of high density lipoproteins (HDL)-cholesterol levels (7-14 mg/dl) was detected in three members (father, son, and daughter) of an Italian family. The three affected individuals did not show any clinical signs of atherosclerosis, nor was the atherosclerotic disease significantly present in the family. Lipoprotein lipase and lecithin:cholesterol acyltransferase activites were normal or slightly reduced. Morphological and compositional studies of HDL in the subjects showed a significant enlargement of the lipoprotein particles (approximately 120 vs. approximately 94 A for control HDL) and a concomitant increase in the triglyceride content. Analytical isoelectric focusing of HDL apoproteins provided evidence for multiple isoproteins in the apoprotein(apo)-A-I range, with nine different bands being detected instead of the usual four bands observed in normal subjects. Two-dimensional immunoelectrophoresis against apo-A antiserum indicated a clear reduction of apo-A in the alpha electrophoretic region, with splitting of the protein peak. The observation in otherwise clinically healthy subjects of hypertriglyceridemia, reduced HDL-cholesterol, and marked apoprotein abnormalities, without a significant incidence of atherosclerotic disease in the family suggests this is a new disease entity in the field of lipoprotein pathology, very probably related to an altered amino acid composition of the apo-A-I protein (see Weisgraber et al. 1980. J. Clin. Invest. 66: 901-907).

Canine Lipoproteins and Atherosclerosis II. Characterization of the Plasma Lipoproteins Associated with Atherogenic and Nonatherogenic Hyperlipidemia

Characterization of the hyperlipoproteinemia induced by feeding high-cholesterol diets to hypothyroid dogs was undertaken in an attempt to identify a lipoprotein pattern or a specific lipoprotein responsible for the atherosclerosis associated with such hyperlipoproteinemia. Various degrees of hyperlipidemia and atherosclerosis were produced during the diet, which was imposed for 3 months to more than a year. Dogs referred to as hyporesponders did not develop significant atherosclerosis despite plasma cholesterol levels ranging from two to five times normal or up to 750 mg/100 ml. This nonatherogenic hyperlipidemia was characterized by an increase in the LDL (low density lipoprotein) and HDL c classes (HDL c refers to a broad spectrum of cholesterol-enriched particles which resemble high density lipoproteins). Dogs referred to as hyperresponders developed significant and often complicated atherosclerosis. Their plasma cholesterol levels were in excess of 750 mg/100 ml, and most of the increased cholesterol was present in lipoproteins with density less than 1.006 g/ml. Several classes of lipoproteins were isolated by ultracentrifugation and purified by Geon-Pevikon block electrophoresis. These lipoproteins were characterized with respect to chemical composition, electrophoretic mobility, immunochemical reactivity, electron microscopic size after negative staining, and apoprotein content as determined by polyacrylamide gel electrophoresis. We concluded that the atherogenic hyperlipidemia of the hyperresponders was characterized by an increase in the amount of cholesterol-rich HDL c as in the hyporesponders but was distinguished by the appearance of a spectrum of cholesterol-rich lipoproteins in the fraction with a density less than 1.006 g/ml. One of these cholesterol-rich lipoproteins had the properties of β very low density lipoproteins, and the others had those of HDL c . We suggest that the spectrum of lipoproteins with density less than 1.006 g/ml represents remnants that accumulate because of defective catabolism of lipoproteins synthesized to carry excess of dietary cholesterol.

A-Imilano apoprotein. Isolation and characterization of a cysteine-containing variant of the A-I apoprotein from human high density lipoproteins.

A recently discovered familial lipoprotein disorder is characterized by reduced plasma levels of high density lipoproteins (HDL) and elevated triglyceride levels. The clinical aspects of this disorder are presented in an accompanying article (Franceschini et al. 1980. J. Clin. Invest. 66: 892-900). The apoprotein content of the HDL isolated from these patients differed markedly from that of normal HDL in that three apoprotein bands not previously described in man were present as major protein components. As determined by sodium dodecyl sulfate (SDS) gel electrophoresis, the relative molecular weights (Mr) of these new apoprotein bands were 55,000, 35,000, and 28,000. Although the Mr 28,000 apoprotein coelectrophoresed with authentic A-I on SDS polyacrylamide gels and showed immunochemical identity with the A-I apoprotein when tested with monospecific apo-A-I antiserum, it contained two amino acid residues, cysteine and isoleucine, which were not present in the amino acid sequence of normal human apo-A-I. This variant form of the A-I apoprotein was designated the A-IMilano apoprotein and denoted A-Icys. By virtue of the presence of cysteine (2 mol/mol A-Icys), the A-Icys apoprotein was capable of forming intermolecular disulfide bonds, and dimer formation of A-Icys produced the Mr 55,000 apoprotein. The Mr 35,000 apoprotein was composed of two different subunits, A-Icys and A-II. By analogy to the apo(E--A-II) complex, which also occurs in human HDL, this mixed disulfide complex was designated as the apo(A-Icys--A-II) complex. The A-IMilano (A-Icys) is the first example of a variation in the primary sequence of a protein of plasma lipoproteins.

Canine Lipoproteins and Atherosclerosis I. Isolation and Characterization of Plasma Lipoproteins from Control Dogs

Canine plasma lipoproteins were fractionated into four distinct classes by ultracentrifugation combined with Geon-Pevikon block electrophoresis and characterized with respect to physical and chemical properties. The distribution of plasma lipids and lipoproteins was quite unlike that in man, the dog having approximately five to six times as much high density as lower density lipoproteins. Despite the marked difference in distribution, human lipoprotein equivalents were present. Very low density lipoproteins (VLDL) isolated at density less than 1.006 g/ml were triglyceride-rich particles ranging in size from 260 to 900 Å in diameter. The density range from 1.006 to 1.063 g/ml contained two classes of lipoproteins: one closely resembled aolow density lipoprotein (LDL) with β mobility and a particle size of approximately 200 Å and the other (called HDL1) was closely related to the high density lipoproteins with respect to immunochemical reactivity, electrophoretic mobility, and apoprotein content. The HDL1 particles ranged in size from 100 to 350 Å in diameter and appeared to be unlike any of the commonly described human lipoproteins. High density lipoproteins called HDL2 isolated in the density range from 1.087 to 1.21 g/ml were protein-rich particles ranging in size from 55 to 85 Å. The apolipoprotein patterns of VLDL, LDL, and HDL2 on polyacrylamide gel electrophoresis were similar to those of the corresponding lipoproteins of man.

Identifying polyglutamine protein species in situ that best predict neurodegeneration

SUMMARY Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function on proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies (IBs), IB formation may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that monoclonal antibody (mAb) 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular weight conformational states expanded polyQ assumes and disappears in higher molecular-weight aggregated forms, such as IBs. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Fat feeding in humans induces lipoproteins of density less than 1.006 that are enriched in apolipoprotein [a] and that cause lipid accumulation in macrophages.

Formula diets containing lard or lard and egg yolks were fed to six normolipidemic volunteers to investigate subsequent changes in the composition of lipoproteins of d less than 1.006 g/ml and in their ability to bind and be taken up by receptors on mouse macrophages. Both formulas induced the formation of d less than 1.006 lipoproteins that were approximately 3.5-fold more active than fasting very low density lipoproteins (VLDL) in binding to the receptor for beta-VLDL on macrophages. Subfractionation of postprandial d less than 1.006 lipoproteins by agarose chromatography yielded two subfractions, fraction I (chylomicron remnants) and fraction II (hepatic VLDL remnants), which bound to receptors on macrophages. However, fraction I lipoproteins induced a 4.6-fold greater increase in macrophage triglyceride content than fraction II lipoproteins or fasting VLDL. Fraction I lipoproteins were enriched in apolipoproteins (apo) B48, E, and [a]. Fraction II lipoproteins lacked apo[a] but possessed apo B100 and apo E. The apo[a] was absent in normal fasting VLDL, but was present in the d less than 1.006 lipoproteins (beta-VLDL) of fasting individuals with type III hyperlipoproteinemia. The apo[a] from postprandial d less than 1.006 lipoproteins was larger than either of two apo[a] subspecies obtained from lipoprotein (a) [Lp(a)] isolated at d = 1.05-1.09. However, all three apo[a] subspecies were immunochemically identical and had similar amino acid compositions: all were enriched in proline and contained relatively little lysine, phenylalanine, isoleucine, or leucine. The association of apo[a] with dietary fat-induced fraction I lipoproteins suggests that the previously observed correlation between plasma Lp(a) concentrations and premature atherosclerosis may be mediated, in part, by the effect of apo[a] on chylomicron remnant metabolism.

Interaction of plasma lipoproteins containing apolipoproteins B and E with heparin and cell surface receptors.

Low density lipoproteins (LDL) containing apolipoprotein B and a high density lipoprotein (the HDLc) containing apolipoprotein E bind to the same cell surface receptors of fibroblasts and also bind to and are precipitated by heparin. To determine if the nature of the chemical interaction of these lipoproteins with the receptor and with heparin were identical, we undertook to compare the effects of the chemical modification of selected amino acid residues of these lipoproteins on receptor and heparin binding activities. Previously, we reported the importance of the positively charged amino acids arginine and lysine in the interaction of LDL and HDLc with the cell surface receptors. Lysine residues were modified by a procedure which did not alter the positive charge on the ϵ-amino group (reductive methylation) or by procedures which neutralized this positive charge (acetoacetylation and carbamylation). Modification of arginyl residues was accomplished using 1,2-cyclohexanedione. Whereas all procedures used to modify the arginine and lysine residues totally abolished receptor binding activity, only the procedures which neutralized the positive charge of these residues abolished heparin binding. Reductive methylation of the lysine residues, which preserved the positive charge on the ϵ-amino group, did not significantly prevent heparin binding or heparin precipitation of LDL or HDLc. Several conclusions are possible from the results of these studies. The protein moieties of both the LDL (apolipoprotein B) and HDLc (apolipoprotein E) can interact with heparin. Furthermore, both arginine and lysine residues participate in the interaction of these lipoproteins with heparin,apparently through an ionic interaction between the positively charged guanido group of arginine or the ϵ-amino group of lysine and the heparin. Although the same residues (arginine and lysine) are involved in cell surface receptor binding of these lipoproteins, the nature of the chemical interaction appears to be different and does not appear to be strictly ionic.

Intravenous infusion of apolipoprotein E accelerates clearance of plasma lipoproteins in rabbits.

Plasma cholesterol levels in cholesterol-fed rabbits were markedly reduced by the intravenous infusion or bolus injection of recombinant human apo E or rabbit plasma apo E. Administration of 6-70 mg of apo E resulted in an approximately 20-40% acute reduction in plasma cholesterol levels within 2-3 h. Plasma cholesterol levels remained reduced for 4-8 h after the administration of apo E. Furthermore, the intravenous injection of apo E reduced the plasma cholesterol levels in Watanabe heritable hyperlipidemic rabbits. The addition of apo E to [14C]cholesterol-labeled canine thoracic duct lymph or [14C]cholesterol-labeled chylomicrons resulted in accelerated plasma clearance of these diet-induced lipoproteins in normal rabbits, with the uptake occurring primarily in the liver. This study suggests that the amount or availability of apo E in the plasma of cholesterol-fed rabbits may be rate limiting for the normal clearance of diet-induced remnant lipoproteins.

A novel electrophoretic variant of human apolipoprotein E. Identification and characterization of apolipoprotein E1.

A new apolipoprotein E (apo E) phenotype has been demonstrated in a Finnish hypertriglyceridemic subject (R.M.). At the time of this study, R.M.s plasma triglyceride and cholesterol levels were 1,021 and 230 mg/dl, respectively. The subjects apo E isoelectric focusing pattern was characterized by two major bands, one in the E3 position and the other in the E1 position. Normally the E1 position is occupied by sialylated derivatives of apo E4, E3, or E2. The E1 band of subject R.M. is not a sialylated form, however, because it was not affected by neuraminidase digestion. The identity of the E1 variant as a genetically determined structure was established by amino acid and partial sequence analyses, confirming that the variant is an example of a previously uncharacterized apo E phenotype, E3/1. Both cysteamine modification and amino acid analysis demonstrated that this variant contains two cysteine residues per mole. Sequence analysis of two cyanogen bromide fragments and one tryptic fragment of the apo E3/1 showed that it differs from E2(Arg158----Cys) at residue 127, where an aspartic acid residue is substituted for glycine. This single amino acid interchange is sufficient to account for the one-charge difference observed on isoelectric focusing gels between E2(Arg158----Cys) and the E1 variant. The variant has been designated E1 (Gly127----Asp, Arg158----Cys). When compared with apo E3, the E1 variant demonstrated reduced ability to compete with 125I-LDL for binding to LDL (apo B,E) receptors on cultured fibroblasts (approximately 4% of the amount of binding of apo E3). This defective binding is similar to that of E2-(Arg158----Cys). Therefore, the binding defect of the variant is probably due to the presence of cysteine at residue 158, rather than aspartic acid at residue 127. In contrast, the apo E3 isoform from this subject demonstrated normal binding activity, indicating that it has a normal structure. In family studies, the vertical transmission of the apo E1 variant has been established. It is not yet clear, however, if the hypertriglyceridemia observed in the proband is associated with the presence of the E1(Gly127----Asp, Arg158----Cys) variant.