The disulfide bonding pattern in ficolin multimers.

نویسندگان

  • Tomoo Ohashi
  • Harold P Erickson
چکیده

Ficolin is a plasma lectin, consisting of a short N-terminal multimerization domain, a middle collagen domain, and a C-terminal fibrinogen-like domain. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12-mers. Two cysteine residues in the N-terminal domain are thought to mediate multimerization by disulfide bonding. We have generated three mutants of ficolin alpha in which the N-terminal cysteines were substituted by serines (Cys4, Cys24, and Cys4/Cys24). The N-terminal cysteine mutants were produced in a mammalian cell expression system, purified by affinity chromatography, and analyzed under nondenaturing conditions to resolve the multimer structure of the native protein and under denaturing conditions to resolve the disulfide-linked structure. Glycerol gradient sedimentation and electron microscopy in nondenaturing conditions showed that plasma and recombinant wild-type protein formed 12-mers. The Cys4 mutant also formed 12-mers, but Cys24 and Cys4/Cys24 mutants formed only trimers. This means that protein interfaces containing Cys4 are stable as noncovalent protein-protein interactions and do not require disulfides, whereas those containing Cys24-Cys24 require the disulfides for stability. Proteins were also analyzed by nonreducing SDS-PAGE to show the covalent structure under denaturing conditions. Wild-type ficolin was covalently linked into 12-mers, whereas elimination of either Cys4 or Cys24 gave dimers and monomers. We present a model in which symmetric Cys24-Cys24 disulfide bonds between trimers are the basis for multimerization. The model may also be relevant to collectin multimers.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join thro...

متن کامل

Human L-Ficolin (Ficolin-2) and Its Clinical Significance

Human L-ficolin (P35, ficolin-2) is synthesised in the liver and secreted into the bloodstream where it is one of the major pattern recognition molecules of plasma/serum. Like other ficolins, it consists of a collagen-like tail region linked to a fibrinogen-related globular head; a basic triplet subunit arises via a collagen-like triple helix, and this then forms higher multimers (typically a 1...

متن کامل

DBCP: a web server for disulfide bonding connectivity pattern prediction without the prior knowledge of the bonding state of cysteines

The proper prediction of the location of disulfide bridges is efficient in helping to solve the protein folding problem. Most of the previous works on the prediction of disulfide connectivity pattern use the prior knowledge of the bonding state of cysteines. The DBCP web server provides prediction of disulfide bonding connectivity pattern without the prior knowledge of the bonding state of cyst...

متن کامل

Disulfide Bonding Pattern Prediction Using Support Vector Machine with Parameters Tuned by Multiple Trajectory Search

The prediction of the location of disulfide bridges helps towards the solution of protein folding problem. Most of previous works on disulfide connectivity pattern prediction use the prior knowledge of the bonding state of cysteines. In this study an effective method is proposed to predict disulfide connectivity pattern without the prior knowledge of cysteins’bonding state. In previous research...

متن کامل

Prediction of Disulfide Bonding Pattern Based on Support Vector Machine with Parameters Tuned by Multiple Trajectory Search

The prediction of the location of disulfide bridges helps solving the protein folding problem. Most of previous works on disulfide connectivity pattern prediction use the prior knowledge of the bonding state of cysteines. In this study an effective method is proposed to predict disulfide connectivity pattern without the prior knowledge of cysteins’bonding state. To the best of our knowledge, wi...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • The Journal of biological chemistry

دوره 279 8  شماره 

صفحات  -

تاریخ انتشار 2004