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T J Knott

Publications and source records attributed to T J Knott.

At least 19 recordsLinked to original sources
PubMed1997

Amino terminus of apolipoprotein B suffices to produce recognition of malondialdehyde-modified low density lipoprotein by the scavenger receptor of human monocyte-macrophages.

Malondialdehyde, a product of lipid peroxidation, produces threshold conversion of low density lipoprotein (LDL) to a form recognized by type I and type II scavenger receptors of monocyte-macrophages. To investigate whether localized domains of human apoB-100 protein provide recognition determinants, we tested the ability of several different apoB-bearing particles to interact with the scavenger receptor of human monocyte-macrophages. Genetically engineered, carboxyl-terminally truncated apoB proteins assembled into lipoprotein form were labeled by fluorescent dye. Fluorescence microscopy and quantitative fluorescent spectrophotometry showed that purified particles containing as little as 23% of the apoB amino-terminus were internalized by the scavenger receptor after, but not before, malondialdehyde modification. There was no recognition of the particles by the LDL receptor. Similar results were obtained with human plasma LDL homozygous for carboxyl-terminally truncated apoB-45.2. Liposome-incorporated fusion protein containing apoB residues 547-735 displayed specific uptake by the scavenger receptor without modification by malondialdehyde. In contrast, fusion proteins containing apoB residues 3,029-3,133 or a short amino terminal segment failed to interact. Thus, primary sequence presented by residues 1-1,084 sufficed to produce recognition of modified LDL by the scavenger receptor. These receptor-combining domains were sequestered when secreted in lipoprotein form and were expressed upon malondialdehyde modification. When packaged exogenously in liposome form, fusion protein containing apoB residues 547-735, containing approximately 4% of the primary sequence, mediated scavenger receptor-dependent uptake and hydrolysis. Our findings provide an additional function or the amino-terminal region of apoB and demonstrate that primary sequence presented by the first 2% of apoB-100 protein suffices to produce recognition on malondialdehyde-modified LDL by the scavenger receptor of human monocyte-macrophages.

Amino Acid Sequence↗
PubMed1991

Sequence requirements for the editing of apolipoprotein B mRNA.

Apolipoprotein (apo) B48 is produced in the mammalian intestine by a tissue-specific RNA-editing mechanism, which mediates a C to U conversion at position 6666 in apoB mRNA. This generates an inframe translation stop codon (UAA) in place of glutamine (CAA) at position 2153. To establish the sequences required for editing we have used an in vitro conversion assay to monitor the editing of synthetic RNAs by rat intestinal extracts. Transcripts containing 55 nucleotides (positions 6649-6703) or more of human apoB mRNA sequence were edited in vitro. Transcripts containing 42 nucleotides (positions 6648-6689) and 26 nucleotides (positions 6662-6687) were edited at 62 and 24% efficiency, respectively, of the 55-nucleotide sequence. To delineate the precise sequence requirements for editing, mutants were generated where 6-nucleotide sections of the 55-base region were changed to anti-sense sequence. Mutation of the 12-nucleotide region immediately downstream of C-6666 abolished editing, and mutation of 6-base sequences immediately 3' and 5' of this 12-nucleotide region significantly reduced editing. Having identified the key region of interest, a panel of 46 mutant RNAs carrying single base substitutions or deletions between nucleotide positions 6657 and 6685 was constructed. Mutagenesis in the sequence 5'-TGATCAGTATA-3' (positions 6671-6681) downstream of C-6666 had the most dramatic effect, since almost all mutations abolished or greatly reduced conversion in vitro. These results suggest that editing is a highly sequence-specific process. We propose that this downstream region is a recognition and/or binding site for the editing enzyme. A search for this sequence in other genes may help to reveal other RNAs that undergo editing.

Animals↗
PubMed1991

Carboxyl-terminal truncation of apolipoprotein B results in gradual loss of the ability to form buoyant lipoproteins in cultured human and rat liver cell lines.

Apolipoprotein B has an obligatory role in the production of chylomicrons, VLDL, and LDL. Familial hypobetalipoproteinemia is a codominant disorder characterized by reduced levels of apo B containing lipoproteins in plasma. We have previously described mutations of the apo B gene in persons with hypobetalipoproteinemia that predict truncated forms of apo B designated apo B29 (1305 amino acid residues) and apo B39 (1799 residues). Apo B39 was present in the VLDL and LDL fractions of plasma, but apo B29 was not detected in the lipoprotein or infranatant fractions of plasma. Here we have investigated the regions of apo B necessary for apo B containing lipoprotein secretion by expression of constructs designed to express truncated forms of apo B. Apo B13 (583 residues), apo B17 (784 residues), apo B23 (1084 residues), apo B29 (1306 residues), and apo B41 (1880 residues) were transiently expressed in HepG2 cells, and apo B23 and apo B41 were stably expressed in McArdle 7777 cells. Lipoprotein (d less than 1.25 g/mL) and infranatant (d greater than 1.25 g/mL) fractions of conditioned medium were analyzed by immunoprecipitation and SDS-PAGE. The distribution between lipoprotein and infranatant fractions varied: apo B41 was found solely in the lipoprotein fraction; apo B29, apo B23, and apo B17 were present in both fractions, but with stepwise truncation, progressively more apo B was recovered in the infranatant; apo B13 was only in the infranatant. These results demonstrate that deletion from the carboxyl terminal of apo B41 results in a gradual loss of the ability of the truncated proteins to form buoyant lipoprotein particles.

Animals↗
PubMed1991

An additional editing site is present in apolipoprotein B mRNA.

Human intestinal apolipoprotein (apo) B mRNA undergoes a C to U RNA editing at nucleotide 6666 to generate a translation stop at codon 2153, which defines the carboxy-terminal of apo B48. Here we show that two of eleven human intestinal cDNAs spanning residue 6666 were edited from a genomically-encoded C to a T at residue 6802 as well as at residue 6666. This additional editing converts Thr (ACA) codon 2198 to Ile (AUA). Synthetic RNA including the nucleotide 6802 was edited in vitro by intestinal extracts at 10-15% of the editing efficiency of nucleotide 6666. A sequence is identified as important for recognition by the editing activity. No secondary structural homology was identified between the two edited sites. No other sequence in the region between 6411 and 6893 nucleotides of apo B mRNA was found to be edited in vivo or in vitro. Apo B RNA editing extracts from intestine did not edit maize cytochrome oxidase II mRNA.

Apolipoproteins B↗
PubMed1988

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.

Amino Acid Sequence↗
PubMed1988

Regional chromosomal localisation of APOA2 to 1q21-1q23.

Using in situ hybridisation, we have mapped APOA2 to the 1q21-1q23 region of chromosome 1. DNA hybridisation to somatic cell hybrids made from cells carrying a balanced translocation between X and 1 confirms the localisation as proximal to 1q23. This was further confirmed by the presence of two polymorphic alleles in a cell line carrying a deletion of 1q25-1q32.

Animals↗
PubMed1988

Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: alternative exon usage predicts two different precursor proteins.

The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.

Amino Acid Sequence↗
PubMed1988

Apolipoprotein B: a novel mechanism for deriving two proteins from one gene.

Evidence from antibody and peptide mapping, protein sequencing and amino acid composition studies suggests that intestinal apo-B48 is colinear with the amino terminal half of hepatic apo-B100. To investigate the mechanism of apo-B48 production we examined cDNA clones from human and rabbit small intestine. All clones contained a single C----T base difference from the hepatic sequence resulting in a translational stop at codon 2153. Amplification by the polymerase chain reaction of cDNA from human and rabbit small intestine, rabbit liver and the human hepatoma cell line HepG2 showed that the stop codon was only present in intestinal mRNA. Enterocyte genomic DNA did not contain the stop codon. We suggest that a co- or post-transcriptional C----U change may result in the production of apo-B48, which represents the amino terminal 2152 amino acids of apo-B100. This is the first example of tissue specific modification of a single mRNA nucleotide resulting in two different proteins from the same primary transcript.

Animals↗
PubMed1987

A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine.

Evidence suggests that intestinal apo-B48 is colinear with the amino-terminal half of hepatic apo-B100. To investigate the mechanism of apo-B48 production, we examined cDNA clones from human and rabbit small intestine. All clones contained a single C----T base difference from the hepatic sequence, resulting in a translational stop at codon 2153. Amplification by the polymerase chain reaction of cDNA from human and rabbit small intestine, rabbit liver, and the human hepatoma cell line HepG2 showed that the stop codon was only present in intestinal mRNA. Enterocyte genomic DNA did not contain the stop codon. We suggest that a co- or posttranscriptional C----U change may result in the production of apo-B48, which represents the amino-terminal 2152 amino acids of apo-B100. This is the first example of tissue-specific modification of a single mRNA nucleotide resulting in two different proteins from the same primary transcript.

Amino Acid Sequence↗
PubMed1987

Expression of multiple growth factors in a human lung cancer cell line.

U-1810, a human large-cell lung cancer line, was found to express a PDGF-like growth factor. 35S-cysteine labelling and immunoprecipitation revealed the synthesis and secretion of a 31-kDa PDGF-like protein. Serum-free conditioned medium contained PDGF-receptor-competing and mitogenic activity when tested on human fibroblasts. Whereas the receptor-competing activity was fully neutralized by anti-PDGF antibodies, the mitogenic activity was only partially affected. We therefore probed U-1810 mRNA with a panel of growth-factor DNA clones. We found expression of the genes for PDGF A- and B-chains, TGF-alpha, TGF-beta and IGF-II but not EGF or IGF-I. U-1810 cells lacked specific binding sites for PDGF but showed specific binding of EGF and expressed EGF-receptor transcripts. Thus, U-1810 is an example of a human tumor cell line that expresses multiple growth factor genes; in the intact tumor the corresponding growth factors may operate in autocrine stimulation of the tumor cells as well as in paracrine growth reactions (i.e. stroma recruitment).

Cell Line↗
PubMed1986

Structure of the human apolipoprotein B gene.

Human apolipoprotein B100 cDNA is 14 kilobases in length and encodes a 4563-amino acid precursor protein. The corresponding human gene has been isolated as a series of overlapping lambda clones and extends over 43 kilobases. The gene comprises 29 exons and 28 introns. The distribution of introns is extremely asymmetrical, most of them appearing in the 5'-terminal one-third of the gene. Although most of the exons fall within the normal size limits for mammalian genes, two are unusually long: 1906 and 7572 base pairs. The latter exon is by far the longest reported for a vertebrate gene.

Apolipoproteins B↗
PubMed1986

Common DNA polymorphism within coding sequence of apolipoprotein B gene associated with altered lipid levels.

60 of 83 middle-aged white men had an XbaI restriction site polymorphism within the coding sequence of the apolipoprotein B gene. Subjects homozygous and heterozygous for the presence of an XbaI restriction site had mean serum triglyceride levels 36% higher (p = 0.02) than those in homozygotes without the restriction site; there was a less substantial difference (p = 0.03) in serum cholesterol. The findings supported a dominant pattern of inheritance. The presence of this restriction site may increase the risk of atherosclerotic disease.

Apolipoproteins B↗
PubMed1986

Regional mapping of human chromosome 19: organization of genes for plasma lipid transport (APOC1, -C2, and -E and LDLR) and the genes C3, PEPD, and GPI.

We report the regional mapping of human chromosome 19 genes for three apolipoproteins and a lipoprotein receptor as well as genes for three other markers. The regional mapping was made possible by the use of a reciprocal whole-arm translocation between the long arm of chromosome 19 and the short arm of chromosome 1. Examination of three separate somatic cell hybrids containing the long arm but not the short arm of chromosome 19 indicated that the genes for apolipoproteins CI, CII, and E (APOC1, APOC2, and APOE, respectively) and glucose-6-phosphate isomerase (GPI) reside on the long arm, whereas genes for the low density lipoprotein receptor (LDLR), complement component 3 (C3), and peptidase D (PEPD) reside on the short arm. When taken together with previous studies, our results suggest the following physical gene map: pter-LDLR-C3-p13.2-PEPD-centromere-(APOE, APOC1, APOC2, GPI)-qter. In addition, we have isolated a single lambda phage carrying both APOC1 and part of APOE. These genes are tandemly oriented and are separated by about 6 kilobases of genomic DNA. Since previous family studies indicate tight linkage of APOE and APOC2, the apolipoprotein genes APOC1, APOC2, and APOE form a tight complex on the long arm of chromosome 19, suggesting the possibility of coordinate regulation.

Apolipoprotein C-I↗