Molecular characterization of Limulus polyphemus C-reactive protein. I. Subunit composition

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• C-reactive protein (CRP) is a key component of the innate immune system.
• Vertebrate CRPs, known as pentraxins, typically assemble from pentameric subunits.
• The structure and subunit composition of Limulus polyphemus CRP were previously less understood.

Purpose of the Study:

• To precisely determine the molecular mass of native C-reactive protein (CRP) from Limulus polyphemus.
• To characterize the subunit composition and glycoforms of Limulus CRP.
• To quantify the concentration of Limulus CRP in hemolymph.

Main Methods:

• Analytical ultracentrifugation was used to determine the molecular mass of native Limulus CRP.
• Electrospray mass spectrometry was employed to analyze dissociated Limulus CRP subunits and identify glycoforms.
• Specific electroimmunoassay was utilized to measure CRP concentrations in hemolymph samples.

Main Results:

• The apparent weight-average molecular mass of native Limulus CRP was determined to be 300 kDa.
• Electrospray mass spectrometry revealed nine components in the dissociated CRP, corresponding to 12 subunits with various glycosylation patterns.
• Limulus CRP concentrations in hemolymph varied widely (0.275–6.64 mg/ml), with a mean of 1.83 mg/ml.

Conclusions:

• Limulus polyphemus CRP is a large, multimeric protein composed of 12 subunits, differing from the pentameric structure of vertebrate CRPs.
• The study identified novel glycoforms of Limulus CRP subunits, expanding the known molecular characterization of this protein.
• Further research is needed to determine if Limulus CRP functions as an acute-phase reactant, similar to its vertebrate homologs.