Apolipoprotein C-II deficiency associated with nonfunctional mutant forms of apolipoprotein C-II

Gf, Maguire; Ja, Little; Kakis, G.; Wc, Breckenridge

doi:10.1139/o84-108

Cited by 43 publications

(10 citation statements)

References 2 publications

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“…We previously reported that the 14 homozygotes and 23 obligate heterozygotes from the apoCII-deficient family described by this laboratory are characterized by the presence of a mutant, nonfunctional apoCII, apoCIITOrmftO (apoCIIT) (11).…”

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confidence: 99%

Apolipoprotein CIISt. Michael. Familial apolipoprotein CII deficiency associated with premature vascular disease.

Connelly¹,

Maguire²,

Little³

1987

J. Clin. Invest.

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A 60-yr-old woman and her brother, products of a consanquinous mating, were chylomicronemic. The chylomicronemia in both subjects was found to be due to the absence of functional apoCI. A mutant form, designated apoClIst. Michael (apo-CIIS), was identified by two-dimensional electrophoresis and Western blot using anti-apoCII antiserum. The isoelectric point of apoCIls was similar to that of normal apoClI, but its apparent molecular weight was 3,000 greater. Tryptic peptides of apoCIIs were identified that had retention times in reversephase high pressure liquid chromatography and amino acid compositions indistinguishable from that of residues 1 to 48 and 51 to 55 of normal apoCI. The complete sequence of apoClIs was deduced fromn a combination of the sequence analysis of tryptic peptides corresponding to residues 56 through 96 and the known sequence of the apoCII gene. ApoCIIs differed from apoCHI at residue 70 where Gln70 was replaced by Pro70 and the sequence terminated with Pro6. This is consistent with a base insertion in the codon for Asp69 or Gln70 in the apoCII gene and a subsequent translation reading frame shift. Both patients were homozygous for apoE4. This and the absence of normal apoCII is consistent with homozygozity at the apoE-CII gene locus on chromosome 19. Both siblings and several relatives had premature ischemic vascular disease, in contrast with its apparent absence in other apoCII-deficient families.

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confidence: 99%

Apolipoprotein CIISt. Michael. Familial apolipoprotein CII deficiency associated with premature vascular disease.

Connelly¹,

Maguire²,

Little³

1987

J. Clin. Invest.

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“…The lyophilized protein was delipidated with ethanol/diethylether (11). The analysis of this acetone-soluble product by analytical polyacrylamide isoelectric focusing using a pH 4-6.5 gradient showed it to contain apoC-IIT and the isomorphs of apolipoprotein C-IIIi.e., C-III-0, C-III-1, and C-III-2 (8).…”

Section: Methodsmentioning

confidence: 98%

“…The viscous oil-like residue present was extracted with 5 ml of chloroform/methanol (2:1) at room temperature to produce a white powder. ApoC-IIT was identified by analytical isoelectric focusing and shown to be homogeneous (8). Normal apoC-II was isolated from a control subject following the identical protocol.…”

Section: Methodsmentioning

confidence: 99%

“…Sodium iodoacetate (0.5 M, pH 8.0) was then added to a final concentration of 0.1 M and the samples were incubated at room temperature for an additional 30 min. The samples were then evaluated for the incorporation of a negative charge by analytical isoelectric focusing (8,14).…”

Section: Methodsmentioning

confidence: 99%

“…Their plasma contains a unique apoC-II that has an apparent pI of 5.54 instead of 4.88 as in normal apoC-II. It was identified as a mutant apoC-II by immunoblot analysis with an antiserum specific for apoC-II (8). In this publication, we describe the purification and the primary structure of this mutant apoC-II, which we designate apoC-IIToronto (apoC-IIT).…”

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Structure of apolipoprotein C-IIToronto, a nonfunctional human apolipoprotein.

Connelly¹,

Maguire²,

Hofmann³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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We previously reported a family with apolipoprotein C-II (apoC-II) deficiency characterized by the presence of a mutant apoC-il, apoC-llT0r~0t. We now report the purification and primary structure of apoC-IITr..t. The sequence of apoC-llTrOft. is identical to that of normal apoC-il from residues 1-68. It differs from residue 69, where AspGln7O-Val7l-Leu72_Ser73-Val74-Leu75-Lys76-Gly77-Glu78 Glu79 is replaced by Thr69-Lys70-phe7l-Phe72-Leu73-Cys74. This is consistent with the deletion of a nucleotide in the codon for either Thr'8 (9) to allow the isolation of chylomicrons and very low density lipoproteins from 700 ml of plasma in a single run. The Ti 15 rotor was brought to 3000 rpm and loaded sequentially with 700 ml of 0.15 M NaCl (1.006 g/ml), 700 ml of plasma adjusted with KBr to 1.063 g/ml, and 300 ml of 0.15 M NaCl adjusted with KBr to 1.25 g/ml (solution A). The chylomicrons were floated by centrifugation at 20,000 rpm for 1 hr and, after slowing the rotor to 3000 rpm, they were displaced with 60 ml of solution A. The plasma remaining in the rotor was spun at 30,000 rpm for 18 hr, the rotor was slowed to 3000 rpm, and the very low density lipoproteins were displaced with 50 ml of solution A. Chylomicrons and very low density lipoproteins adhering to the rotor core were isolated separately after the rotor had come to a complete stop. Each fraction was dispersed in 0.15 M NaCl/0.01% Na2EDTA/0.02% NaN3 and reisolated by centrifugation in an 80 Ti rotor for 16 hr at 45,000 rpm.The C apolipoproteins were isolated from these fractions by treatment with acetone (10, 11). The lyophilized protein was delipidated with ethanol/diethylether (11). The analysis of this acetone-soluble product by analytical polyacrylamide isoelectric focusing using a pH 4-6.5 gradient showed it to contain apoC-IIT and the isomorphs of apolipoprotein C-IIIi.e., C-III-0, C-III-1, and C-III-2 (8).ApoC-IIT was purified to homogeneity by a single preparative flat-bed isoelectric focusing step using Ultradex granulated gel and pH 4-6 ampholines (LKB). Approximately 50 mg of acetone-soluble protein was dissolved in 3 ml of 1% decyl sodium sulfate (NaDecSO4)/0.01 M TrisHCl, pH 8.2/10 mM dithiothreitol, applied at the cathodic end of the gel and electrophoresed at 8 W for 20 hr at 12°C in a Hoeffer Isobox apparatus (12). The gel containing apoC-IIT was removed and eluted first with 1 ml and then with 2.5 ml of 0.5% NaDecSO4/0.01 M Tris HCl, pH 8.2. The combined eluates were mixed with ice-cold acetone (9 vol of acetone to 1 vol of eluate) and allowed to stand at 4°C for 10 min. The precipitated protein was pelleted by centrifugation at 2000 rpm for 20 min. The precipitate was washed twice with 10 ml of ice-cold acetone. The viscous oil-like residue present was extracted with 5 ml of chloroform/methanol (2:1) at room temperature to produce a white powder. ApoC-IIT was identified by analytical isoelectric focusing and shown to be homogeneous (8). Normal apoC-II was isolated from a control subject following the identical protocol.Amino iT...

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Studies on an apolipoprotein C‐II variant occurring in Caucasians

Kurth

Oette

1992

Electrophoresis

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Apolipoproteins C (apo C-II, apo C-III0, apo C-III1 and apo C-III2) from delipidated very low density lipoproteins (VLDL) of 522 normo- and hyperlipoproteinemic Caucasians were screened by analytical isoelectric focusing. The immobilized pH gradient used was pH 4.0-5.0 with 7 M urea, which raised the apparent pH range to 4.8-5.7. As identified by immunoblotting, six unrelated persons had two major isoforms of apo C-II, the normal apo C-II-1 (which focuses between apo C-III0 and apo C-III1) and a variant, designated apo C-II-v according to Huff et al., focusing between apo C-III1 and apo C-III2 due to a more acidic pI. In narrow pH gradients, apo C-II-v can readily be discriminated from the minor isoform, apo C-II-2, due to its slightly more basic pI, corresponding to a difference of 0.01 pH units. Neuraminidase treatment did not alter the pI of apo C-II-v and on two-dimensional electrophoresis the molecular weights of apo C-II-1 and apo C-II-v were indistinguishable. The frequency of apo C-II-v was 1.2%. It was the same in males and females and was independent of hypertriglyceridemia. The autosomal codominant inheritance could be demonstrated in the pedigree of one family. Electroblotting of apo C-II-1 and apo C-II-v onto activated glass fiber sheets, followed by amino acid sequence analysis of the amino terminal ends, revealed an exchange of the amino acid lysine at position 19 by threonine in apo C-II-v.(ABSTRACT TRUNCATED AT 250 WORDS)

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Apolipoprotein C-II deficiency associated with nonfunctional mutant forms of apolipoprotein C-II

Cited by 43 publications

References 2 publications

Apolipoprotein CIISt. Michael. Familial apolipoprotein CII deficiency associated with premature vascular disease.

Apolipoprotein CIISt. Michael. Familial apolipoprotein CII deficiency associated with premature vascular disease.

Structure of apolipoprotein C-IIToronto, a nonfunctional human apolipoprotein.

Studies on an apolipoprotein C‐II variant occurring in Caucasians

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