Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains.

Ortel, Thomas L.; Takahashi, Nobuhiro; Putnam, Frank W.

doi:10.1073/pnas.81.15.4761

Cited by 95 publications

(49 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1F, which was typically obtained from samples of rHCp purified at the longer induction times and/or higher cellular density in an attempt to raise the yield of the recombinant protein. The fragmentation pattern now reproduced that usually found in HCp (28), with fragments at ϳ116, 19, and 50 kDa, produced by the cleavage of the polypeptide chain at Lys 887 . Catalytic and Spectroscopic Properties of Recombinant Ceruloplasmin-Optical and EPR spectroscopies were used to analyze the state of copper sites (Fig.…”

Section: Expression Of Human Ceruloplasmin In P Pastoris-expres-supporting

confidence: 74%

“…This is a long recognized peculiarity of Cp that has been outlined by numerous spectroscopic and catalytic studies and it has been confirmed by the x-ray structural studies on the human protein (23). Human ceruloplasmin (HCp) is a single chain of 1046 amino acids (28), with a carbohydrate content of 7-8% and a copper content of six integral copper atoms. The x-ray structure has shown that the molecule of HCp is composed of six compact domains, with large loop insertions, and that the six integral copper atoms are distributed in one trinuclear cluster, located at the border between domains 1 and 6 and possessing ligands from each domain and in three mononuclear T1 sites.…”

Section: Ceruloplasmin (Cp)mentioning

confidence: 80%

See 1 more Smart Citation

Site-directed Mutagenesis of Human Ceruloplasmin

Bielli¹,

Bellenchi²,

Calabrese³

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.

show abstract

Section: Expression Of Human Ceruloplasmin In P Pastoris-expres-supporting

confidence: 74%

Section: Ceruloplasmin (Cp)mentioning

confidence: 80%

Site-directed Mutagenesis of Human Ceruloplasmin

Bielli¹,

Bellenchi²,

Calabrese³

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…2,3 The A and C domains are 40% identical between FV and FVIII, but there is little sequence homology between the B domains. The A domains are similar to the A domains of ceruloplasmin, 4 and the C domains are similar to phospholipid-binding proteins. The crystal structures of the FV and FVIII light-chain C 2 domains have been determined and show hydrophobic and electrostatic interactions important for anchoring to the phospholipid membrane.…”

Section: Introductionmentioning

confidence: 85%

Structural requirements for the activation of human factor VIII by thrombin

Myles

Yun

Leung

2002

Blood

View full text Add to dashboard Cite

The coagulation factors V (FV) and VIII (FVIII) are important at sites of vascular injury for the amplification of the clotting cascade. Natural variants of these factors frequently lead to severe bleeding disorders. To understand the mechanisms of activation of FVIII by thrombin, we used a bank of mutant thrombins to define residues important for its activation. From the initial screening of 53 mutant thrombins for the activation of human recombinant FVIII, we mapped thrombin mutants with 50% or less activity to anion-binding exosite-I (Lys21Ala, His66Ala, Lys65Ala, Arg68Ala, Arg70Ala, and Tyr71Ala) and anion-binding exosite-II (Arg98Ala), the Na ؉ -binding site (Glu229Ala, Arg233Ala, Asp234Ala, and Asp193Ala/Lys196Ala), and the 50-insertion loop (Trp50Ala), which were similar to our results for the activation of FV. The role of these residues for cleavage at Arg372 and Arg1689 was investigated using plasma FVIII. Anion-binding exosite-I appears to be important for cleavage at both sites, whereas the anion-binding exosite-II residue Arg98Ala is important for cleavage at Arg372 alone. The Glu229Ala mutant, which contributes to the Na ؉ -binding site, and the 50-insertion loop mutant W50A have severely impaired cleavage at Arg372 and Arg1689. This suggests that the integrity of the active site and the Na ؉ -bound form of thrombin are important for its procoagulant activity against FVIII. Detailed mutagenic analysis of thrombin can assist in understanding the pathogenesis of bleeding disorders and may lead to the rational design of selective thrombin inhibitors. (Blood. 2002;100:2820-2826)

show abstract

“…The 1 15-kD fragment has been observed in most preparations ofceruloplasmin (unless it is further degraded to 50-and 67-kD fragments) (37,38). Ceruloplasmin prepared by this procedure was completely stable for prolonged periods (> 1 mo at 370C) due to the removal ofan endogenous metalloproteinase that specifically cleaved intact, 1 32-kD ceruloplasmin into 1 Lipoprotein methods. Human LDL was prepared from freshly drawn, citrated normolipemic plasma to which EDTA was added before ultracentrifugation.…”

Section: Methodsmentioning

confidence: 97%

“…It is a monomer of 132 kD composed almost entirely of three 42-45-kD domains that are highly homologous to each other (1) and to the three do-A portion of this work has appeared in abstract form ( 1993. Circula-mains that make up the large, active proteolytic subunits of factors V and VIII of the coagulation cascade (2).…”

Section: Introductionmentioning

confidence: 99%

Intact human ceruloplasmin oxidatively modifies low density lipoprotein.

Ehrenwald¹,

Chisolm²,

Fox³

1994

J. Clin. Invest.

244

153

View full text Add to dashboard Cite

Ceruloplasmin is a plasma protein that carries most of the copper found in the blood. Although its elevation after inflammation and trauma has led to its classification as an acute phase protein, its physiological role is uncertain. A frequently reported activity of ceruloplasmin is its ability to suppress oxidation of lipids. In light of the intense recent interest in the oxidation of plasma LDL, we investigated the effects of ceruloplasmin on the oxidation of this lipoprotein. In contrast to our expectations, highly purified, undegraded human ceruloplasmin enhanced rather than suppressed copper ion-mediated oxidation ofLDL. Ceruloplasmin increased the oxidative modification of LDL as measured by thiobarbituric acid-reacting substances by at least 25-fold in 20 h, and increased electrophoretic mobility, conjugated dienes, and total lipid peroxides. In contrast, ceruloplasmin that was degraded to a complex containing 115-and 19-kD fragments inhibited cupric ion oxidation of LDL, as did commercial preparations, which were also degraded. However, the antioxidant capability of degraded ceruloplasmin in this system was similar to that of other proteins, including albumin. The copper in ceruloplasmin responsible for oxidant activity was not removed by ultrafiltration, indicating a tight association. Treatment of ceruloplasmin with Chelex-100 removed one of seven copper atoms per molecule and completely blocked oxidant activity. Restoration of the copper to ceruloplasmin also restored oxidant activity. These data indicate that ceruloplasmin, depending on the integrity of its structure and its bound copper, can exert a potent oxidant rather than antioxidant action on LDL. Our results invite speculation that ceruloplasmin may be in part responsible for oxidation of LDL in blood or in the arterial wall and may thus have a physiological role that is quite distinct from what is commonly believed. (J. Clin. Invest. 1994. 93:1493-1501

show abstract

Structural model of human ceruloplasmin based on internal triplication, hydrophilic/hydrophobic character, and secondary structure of domains.

Cited by 95 publications

References 18 publications

Site-directed Mutagenesis of Human Ceruloplasmin

Site-directed Mutagenesis of Human Ceruloplasmin

Structural requirements for the activation of human factor VIII by thrombin

Intact human ceruloplasmin oxidatively modifies low density lipoprotein.

Contact Info

Product

Resources

About