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A A Protter

Publications and source records attributed to A A Protter.

33 records · Page 2Linked to original sources
PubMed1987

Carboxyl terminal analysis of human B-48 protein confirms the novel mechanism proposed for chain termination.

In this study we have confirmed the presence of a single base difference between intestinal mRNA coding for B-48 and hepatic mRNA coding for B-100, which results in the substitution of a stop codon (UAA) for a glutamine codon (CAA) at a point corresponding to amino acid residue 2153 in the B-100 sequence. Based on this finding, B-48 is predicted to terminate at residue 2152 with the sequence ... Met Ile. To confirm this finding at the protein level, B-48 and B-100 were each digested with cyanogen bromide and the digestion products were analysed for the presence of isoleucine. Isoleucine was found only in cyanogen bromide digests of B-48 confirming that only B-48 terminates with the predicted amino acid sequence ... Met Ile.

Amino Acid Sequence↗
PubMed1987

Structural comparison of human apolipoproteins B-48 and B-100.

In this study we have investigated the structural relationship between human apolipoproteins B-48 and B-100 by comparing protein structure and by comparing nucleotide sequence from intestinal and hepatic cDNA clones. Sequences from intestinal and hepatic cDNA were identical over the entire distance analyzed (7194 bases), which is more than required to code for B-48. The amino-terminal amino acid sequences from intact B-48 and B-100 proteins were also identical over the entire distance analyzed (16 residues). Additional protein homology was evaluated by the combined techniques of peptide mapping and immunoblotting. Purified B-48 and B-100 were each digested with three different endoproteases, and the resulting peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide bands were then detected by silver stain and by Western blotting with antisera against specific regions of B-48 and B-100. The resulting patterns suggest that B-48 is extensively homologous with the amino-terminal portion of B-100. We have identified only four peptides from B-48 (at least one in each digest) that are absent from the parallel digests of B-100. These peptides appear to arise from the ultimate carboxyl terminus of B-48 and appear to be totally homologous with a region located near the center of B-100. Our observations suggest that mature, circulating B-48 is homologous over its entire length (estimated to be between 2130 and 2144 amino acid residues) with the amino-terminal portion of B-100 and contains no sequence from the carboxyl end of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells.

We produced human apolipoprotein A-I (apoA-I) in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with an expression plasmid which placed the human apoA-I gene under the direction of the human metallothionein II gene promoter. Isolation of a clonal cell line resulted in high level expression of apoA-I. Greater than 30% of total protein secreted by these CHO cells was apoA-I, which enabled us to purify apoA-I with a single step purification scheme. As a result, large quantities of apoA-I can be produced and isolated without having to rely on plasma sources. Structural characterization of the recombinant apoA-I showed it to be identical to authentic apoA-I from human serum high density lipoprotein. Furthermore, we demonstrated approximately equal to 90% of the apoA-I secreted by CHO cells is processed, mature protein. A portion of the secreted recombinant apoA-I was associated with lipid and floated at a density approximately equal to 1.10 g/ml. Additional analysis identified the presence of five isoforms of apoA-I in the CHO cell conditioned medium. Processing and post-translational modification of the recombinant apoA-I occurred in the CHO cell cultures in the absence of serum components. We conclude that the human apoA-I produced by CHO cells is identical to circulating, mature apoA-I in humans and that recombinant mammalian expression offers an opportunity to investigate apoA-I processing.

Amino Acid Sequence↗
PubMed1987

Apolipoprotein B: structure, biosynthesis and role in the lipoprotein assembly process.

The complete amino acid sequence of the liver-synthesized apolipoprotein B (apoB) species, apoB 100, has been derived from cloned cDNA. The protein consists of 4536 amino acids (+ a 27 amino acid signal sequence). Cysteine is clustered in the N-terminal 1/10 of the protein, suggesting the presence of a stabilized tertiary structure in this part of the molecule. Three types of structure are suggested to be of importance for the binding of the protein to lipids; (i) hydrophobic sequences with a high probability for beta-sheet structure, (ii) strict amphipathic beta-sheets, and (iii) amphipathic alfa-helices. An apoB 100 molecule is completed within 10-14 min and secreted after approximately 30 min, 1/3 of which is due to the transfer through the endoplasmic reticulum (ER), while 2/3 is spent in the Golgi apparatus. ApoB 100 is co-translationally N-glycosylated and 25% of the oligosaccharide chains is processed in the Golgi compartment. Other posttranslational modifications that have been discussed include covalent acylation and phosphorylation. It has also been suggested that the lipid moiety of the apoB 100 lipoproteins are modified during the passage through the Golgi apparatus. The site of lipoprotein assembly is suggested to be separated from the site of apoB 100 synthesis, and apoB 100 appears to be co-translationally bound to the ER membrane and from this transferred to the ER lumen. Based on these observations a model for the assembly of apoB 100 lipoproteins is discussed in this paper. The intestinal derived apoB species, apoB 48, has a molecular mass of 210 kDa and appears to correspond to the N-terminal 48% of apoB 100. The mechanism by which apoB 48 is formed is still not known. Available data indicate that the protein is formed within the intestinal cells, these data also argue against the possibility that apoB 48 is formed by posttranslational proteolysis of apoB 100. The formation of a separate apoB 48 mRNA by alternative splicing has been suggested, based on the observation of a 7 kb mRNA which corresponds to the 5' portion of the apoB 100 mRNA. However, the most abundant apoB mRNA species found in the intestine have a size that corresponds to that of the apoB 100 mRNA, furthermore the observation that apoB 48 appears to terminate in a 7.5 kb exon that appears to lack alternative splice sites, does not favour the possibility of alternative splicing.

Amino Acid Sequence↗
PubMed1986

Analysis of cDNA clones encoding the entire B-26 region of human apolipoprotein B.

We report the characterization of intestine and liver cDNA clones for human apolipoprotein B (apoB) that map to the 5' end of the mRNA. The protein sequence encoded by the 5011 nucleotides derived from sequence analysis of these clones includes 1643 amino acid residues of the mature protein of Mr 184,000. The amino acid sequence at the amino terminus of B-74 peptide was determined and mapped to residue 1298. The size (Mr 145,700) and amino acid composition of the B-26 region encoded by these clones (including amino acid residues 1-1297) closely match the values obtained from the B-26 peptide. The amino acid sequence of peptide B-100 at the junction of peptides B-26 and B-74 (Phe-Lys decreases- Ser) shows structural homology to the site on human kininogen (Phe-Arg decreases- Ser) that is cleaved by the protease plasma kallikrein. The encoded protein contains five potential N-glycosylation sites and several regions in which the hydroxyamino acids, serine and threonine, are present in high abundance. The protein sequence presented in this report represents approximately 30% of the total B-100 protein and will aid in the characterization of additional cDNA clones.

Amino Acid Sequence↗
PubMed1986

Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B.

A partial cDNA clone for the B-26 region of apolipoprotein B was isolated from an adult human liver DNA library by screening with an oligonucleotide probe derived from amino-terminal protein sequence obtained from purified B-26 peptide. Antisera against a synthetic 17-residue peptide whose amino acid sequence was encoded by the clone cross-reacts with apolipoproteins B-26, B-100, and B-48, but not with B-74. The nucleotide sequence immediately upstream from the amino terminus of B-26 codes for an apparent signal sequence, implying that the B-26 moiety is in an amino-terminal locus in the B-100 protein. That this sequence represents a 5' end region is further supported by primer extension analysis using a fragment of the cDNA clone and by S1 nuclease protection experiments using the corresponding region in a genomic clone.

Amino Acid Sequence↗
PubMed1984

Independent developmental programs for two estrogen-regulated genes.

Measurement of hepatic apolipoprotein II and vitellogenin II messenger RNA during chicken embryogenesis showed that these genes acquire estrogen responsiveness at different stages of development. Sensitive solution hybridization assays with excess complementary DNA showed that apolipoprotein II transcripts were induced to 500 molecules per cell at day 9, whereas induction of vitellogenin II messenger RNA was not found until day 11. Thus, two estrogen regulated genes of common function and coordinately regulated in the adult may be on independent developmental programs.

Animals↗
PubMed1984

Isolation and DNA sequence of full-length cDNA and of the entire gene for human apolipoprotein AI--discovery of a new genetic polymorphism in the apo AI gene.

We have isolated full-length cDNA clones for apolipoprotein AI from a human fetal liver cDNA library made in the vector lambda gt10. One such clone, pBL13AI, was 965 nucleotides long and contained the entire preproapolipoprotein AI sequence, in addition to 86 nucleotides of 5' untranslated region and 75 nucleotides of the 3' untranslated region. The complete structure of this clone is presented. Furthermore, we have obtained a 20-kb genomic fragment from a human genomic library, encompassing the entire apo AI gene. Sequence analysis of the gene shows that the coding region is interrupted by three introns of 197, 187, and 588 bp, respectively. Digestion of the DNA of various individuals with the endonuclease Msp I revealed a new restriction site polymorphism in the apo AI gene.

Apolipoprotein A-I↗
PubMed1984

Isolation and sequence analysis of the human apolipoprotein CIII gene and the intergenic region between the apo AI and apo CIII genes.

We have isolated and characterized the gene for human apolipoprotein CIII, a major component of the triglyceride-rich lipoproteins. Determination of the complete DNA sequence of this gene revealed that the coding sequence is interrupted by three introns, which occur in the 5' noncoding region, in the region encoding the signal sequence and in the region coding for the mature plasma protein, respectively. We present an analysis of the structure of the apo CIII gene and compare it to the gene for apolipoprotein AI. In addition, we present the DNA sequence of the intergenic region lying between the apo AI and the apo CIII genes.

Apolipoprotein A-I↗
PubMed1982

Isolation and characterization of a cDNA clone specific for avian vitellogenin II.

A clone for vitellogenin, a major avian, estrogen responsive egg yolk protein, was isolated from the cDNA library of estrogen-induced rooster liver. Two forms of plasma vitellogenin, vitellogenin I (VTG I) and vitellogenin II (VTG II), distinguishable on the basis of their unique partial proteolysis maps, have been characterized and their corresponding hepatic precursor forms identified. We have used this criterion to specifically characterize which vitellogenin protein had been cloned. Partial proteolysis maps of BTG I and VTG II standards, synthesized in vivo, were compared to maps of protein synthesized in vitro using RNA hybrid-selected by the vitellogenin plasmid. Eight major digest fragments were found common to the in vitro synthesized vitellogenin and the VTG II standard while no fragments were observed to correspond to the VTG I map. A restriction map of the VTG II cDNA clone permits comparison to previously described cDNA and genomic vitellogenin clones.

Animals↗
PubMed1980

Biosynthesis of apolipoprotein B in rooster kidney, intestine, and liver.

After in vitro incubation with radiolabeled amino acids, extracts of various chicken tissues were screened for newly synthesized proteins reactive with antibody to plasma apolipoprotein B (apo B). An immunologically related protein was found in kidney as well as liver and intestine. This kidney protein showed the same mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis as plasma apo B or pulse-labeled apo B made by liver or intestine. Partial proteolysis mapping with porcine elastase or V8 protease revealed nearly identical partial cleavage maps of pulse-labeled apo B from kidney, intestine, and liver. These data show that the kidney is a site of apo B synthesis and suggest that the kidney may be a significant source of plasma lipoproteins. Apo B synthesis was also detected in the distal small intestine and colon as well as in the proximal small intestine. The relative rate of apo B synthesis was 5% of total protein synthesis in the duodenum and decreased progressively to about 1% of protein synthesis in the colon. The synthesis of apo B in the distal small intestine and colon may indicate a similar capacity for dietary fat absorption via lipoprotein particles in all intestinal regions. The large intestine may be an additional source of plasma lipoproteins.

Animals↗
PubMedDate not supplied

Mapping of human apolipoprotein B antigenic determinants.

A minimum of 16 epitopes which provide a group of topographical markers to study the conformation of apolipoprotein (apo) B have been mapped in relation to elements of the sequence of apo B-100. Six of these epitopes are identified by monoclonal antibodies (Mabs) directed against low density lipoprotein (LDL) apo B, while at least 10 others react with Mabs obtained by immunization with delipidated and solubilized apo B. Five epitopes which are also expressed on apo B-48 have been assigned to the thrombolytic fragment T4 on the N-terminal side of apo B-100. None of these five epitopes requires the presence of lipids for its expression, suggesting that the conformation of the T4 region of apo B is more dependent on peptide-chain interactions than on peptide-lipid interactions. Four distinct epitopes have been assigned to the median thrombolytic fragment T3 of apo B-100, all of which require the presence of lipids for their expression; those epitopes closer to the C-terminus of T3 require specific interaction with cholesteryl esters. The same lipid dependence also characterizes a cluster of epitopes mapped to the N-terminal region of fragment T2. The epitopes that are close to the T2/T3 cleavage site and depend on the presence of cholesteryl esters for their expression are also those that react with the Mabs that inhibit the binding of LDL to its receptor. Therefore this region, which in addition contains two sequences with structural homology to the apo E receptor binding domain, probably constitutes a physiologically important receptor binding site for apo B. Finally, four other distinct epitopes which do not require the presence of lipids for their expression have been mapped on T2. In conclusion, the present report presents evidence that the immunochemical analogy of apo B-48 and apo B-100 is on the N-terminal half of apo B-100, whereas the apo B receptor binding domain is localized on the C-terminal half of apo B-100 close to the T2/T3 cleavage site.

Antibodies, Monoclonal↗