Simon W. Law

cDNA cloning of human apoA-I: Amino acid sequence of preproapoA-I

ds-cDNA to human liver apoA-I mRNA has been cloned. Nucleic acid sequence analysis revealed that apoA-I mRNA codes for a precursor apolipoprotein, preproapoA-I, which contains 24 amino acids on the NH2-terminal end of the mature plasma apoA-I. Eighteen amino acids are contained within the hydrophobic prepeptide (Met-Lys-Ala-Ala-Val-Leu-Thr-Leu-Ala-Val-Leu-Phe-Leu-Thr-Gly-Ser-Gln-Ala) followed by a 6 amino acid propeptide (Arg-His-Phe-Trp-Gln-Gln). Our results on human apoA-I are in agreement with the partial sequence of the precursor of rat apoA-I with respect to the length of the precursor sequence, location of the prepeptide, and the presence of an unusual propeptide sequence terminating in a neutral dipeptide Gln-Gln. A detailed analysis of the primary structure of normal human apoA-I mRNA will be essential to our ultimate understanding of the processing of human apoA-I and diseases characterized by molecular defects in apoA-I structure and function.

Identification of a novel in-frame translational stop codon in human intestine apoB mRNA.

Human apolipoprotein (apo) B exists in plasma as two isoproteins designated apoB-100 and apoB-48. ApoB-100 (512 kDa) and apoB-48 (250 kDa) are synthesized by the liver and intestine respectively. Analysis of apoB cDNA clones isolated from a human intestinal cDNA library revealed that the intestinal apoB mRNA contains a new in-frame translational stop codon. This premature stop codon is generated by a single base substitution of a C to T at nucleotide 6538 which converts the codon CAA coding for the amino acid glutamine residue 2153 to an in-frame stop codon TAA. The generation of a stop codon in the intestinal apoB mRNA appears to be tissue specific since it has not been reported in cDNA clones isolated from human liver cDNA libraries which code for the 4536 amino acid apoB-100. A potential polyadenylation signal sequence AATAAA was also identified 390 bases downstream from the new stop codon. The new stop codon in the human intestinal apoB mRNA provides a potential mechanism for the biosynthesis of intestinal apoB-48.

Identification of low density lipoprotein receptor binding domains of human apolipoprotein B-100: A proposed consensus LDL receptor binding sequence of apoB-100

The human liver apoB-100 gene cloned in the lambda gt-11 expression vector expresses fusion proteins reacting with apoB antibodies. A fusion protein induced from a apoB-lambda gt-11 clone reacted with apoB-100 monoclonal antibodies known to block the binding of LDL to the LDL receptor. The fusion protein contains an amino acid sequence domain enriched in positively charged residues which is complementary to the negatively charged amino acids present in the consensus LDL receptor binding domain. This sequence of apoB-100 is proposed as a binding domain for the interaction with the LDL receptor. Comparison of derived amino acid sequences from the entire structure of apoB-100 molecule revealed several similar domains enriched in positively charged amino acids. A consensus sequence of the potential LDL binding domain was identified which contained positively charged amino acids at positions 1, 5 and 8 and a loop of 8-11 amino acids followed by two adjacent positively charged amino acids. These results are interpreted as indicating that there are several potential LDL receptor binding domains in apoB-100.

Human apolipoprotein A-I and C-III genes reside in the p11 → q13 region of chromosome 11

Apolipoprotein (apo) A-I is a major protein of high density lipoproteins (HDL). The gene for apoA-I has been localized to the p11 leads to q13 region of chromosome 11 by filter hybridization analysis of mouse-human hybrid cell cDNAs containing chromosome 11 translocations utilizing a cloned human apoA-I cDNA probe. The known linkage of apoA-I and apoC-III also permitted the simultaneous assignment of the apoC-III gene to the same region on chromosome 11. Comparison with previously established gene linkages on the mouse and human genome suggests that apoA-I + apoC-III may be linked to the esterase A4 and uroporphyrinogen synthase genes which are present on the long arm of human chromosome 11. The localization of the apoA-I + apoC-III genes in the p11----q13 region of chromosome 11 represents a definitive chromosomal assignment of a human apolipoprotein gene, and will now enable more detailed analysis of the geneomic organization and linkages of the apolipoprotein genes.

The human preproapolipoprotein C-II gene: Complete nucleic acid sequence and genomic organization

The complete nucleic acid sequence of human preproapolipoprotein (apo) C‐II has been determined from 2 apoC‐II clones isolated from 2 different human genomic DNA libraries. The cloned fragments were approx. 14 and 18 kb long, and sequence analysis established that the apoC‐II gene consists of 3338 nucleotides containing 3 intervening sequences of 2391, 167, and 298 bases. The first intron is located within the 5‐untranslated region of apoC‐II and contains 4 Alu type sequences. The second intron interrupts the codon specifying amino acid —11 of the apoC‐II signal peptide. The last intron, which contains a 38 bp sequence which is repeated 6 times, interrupts the codon specifying for amino acid +44 of the mature apolipoprotein.

Human plasma proapoa-I: Isolation and amino-terminal sequence

Human apoA-I is synthesized as preproapoA-I, a 267 amino acid precursor apolipoprotein. PreproapoA-I initially undergoes intracellular co-translational proteolytic cleavage into proapoA-I. ProapoA-I is secreted from the cell and was isolated from thoracic duct lymph in the apoA-I1 isoform position. The amino-terminal sequence of proapoA-I isolated from human lymph revealed the presence of 6 additional amino acids, Arg-His-Phe-Trp-Gln-Gln, on the amino-terminal end of apoA-I consistent with the proapoA-I sequence determined by nucleic acid sequence analysis of cloned apoA-I. Our results indicate that proapoA-I is present in human plasma, and undergoes post-translational proteolytic cleavage to mature plasma apoA-I.

Analysis of the apoC-II gene in apoC-II deficient patients

Apolipoprotein C-II (apoC-II), a 79 amino acid protein, is a cofactor for lipoprotein lipase, the enzyme which catalyzes the lipolysis of triglycerides on plasma chylomicrons and VLDL. Patients with apoC-II deficiency have marked elevations in plasma triglycerides, chylomicrons, VLDL, and a type I hyperlipoproteinemia. In order to evaluate the molecular defect in apoC-II deficiency, genomic DNA was analyzed using Southern Blot from 2 independent apoC-II deficient patients and compared to normal controls. Restriction digests of genomic DNA were performed with five different enzymes and the restriction fragments analyzed utilizing a 354 base pair nick-translated apoC-II probe for hybridization following Southern blotting. The restriction fragments varied from 0.8 to 21 Kb, and the pattern with normal DNA was identical to that of the two apoC-II deficient patients. The present study reveals that the apoC-II gene is present in patients with apoC-II deficiency. In addition, no insertional or deletional polymorphism was detected in the apoC-II gene of apoC-II deficient patients.

The human apolipoprotein A-II gene is located on chromosome 1

Apolipoprotein (apo) A-II is a major constituent of high density lipoproteins (HDL). The gene for apoA-II has been localized to the p21----qter region of chromosome 1 in man by Southern blot hybridization analysis of DNA from human-mouse cell hybrids using a cloned human apoA-II cDNA probe. The regional assignment was established using two hybrids carrying a reciprocal translocation involving chromosomes 1 and 2. Comparison with previously established gene loci on chromosomes 1 suggests that apoA-II may reside in a conserved linkage group with renin and peptidase C. On the other hand, apoA-II is not linked to the apoA-I gene, which has been localized previously to chromosome 11.

Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A‐I mRNA in Hep G2 cells

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A‐I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA‐I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA‐I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA‐I gene expression and regulation.

Human ApoB-100 gene resides in the p23----pter region of chromosome 2.

Human apolipoprotein (apo) B-100 is the major apolipoprotein of low density lipoproteins (LDL) and the principal ligand for interaction with the LDL receptor. The gene for apoB-100 has been localized to the p23----pter region of chromosome 2 by filter hybridization analysis with radiolabelled apoB-100 cDNA probes and human-mouse somatic cell hybrids containing chromosome 2 translocations. Other genes at the end of the short arm of chromosome 2 include acid phosphatase, proopiomelanocortin complex, malate dehydrogenase, and N-myc, the latter gene has been previously localized to the same bands (2p23----pter) as the apoB-100 gene. The localization of the apoB-100 gene to the p23----pter region of chromosome 2 completes the genomic organizational relationship of the LDL receptor and the two apolipoprotein ligands for the LDL receptor, apoE and apoB-100; the LDL receptor and apoE having been previously localized to chromosome 19.