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Quantification of the pro-form of human complement component factor D (adipsin)
Maiken Lumby Henriksen1, Christian Nielsen2, Dennis Pedersen3
1Dept. of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
Insights
A new ELISA assay quantifies pro-factor D, revealing 8-15% of this complement protein circulates in its precursor form. Blood collection methods impact measured levels.
Area of Science:
- Immunology
- Biochemistry
- Complement System Biology
Background:
- Factor D (adipsin) is a serine protease crucial for complement system activation, particularly the alternative pathway.
- It cleaves factor B, forming the C3 convertase C3bBb, central to complement amplification.
- Adipocytes produce factor D as a pro-form, requiring cleavage by MASP-3 to become mature and active.
Purpose of the Study:
- To develop and validate an ELISA for quantifying the pro-form of complement factor D.
- To determine the circulating levels of pro-factor D in healthy individuals.
- To investigate the influence of blood sampling procedures on factor D conversion.
Main Methods:
- Development and validation of a specific ELISA for pro-factor D.
- Assay validation included working range, intra/inter-assay coefficients of variation (CVs), and recovery rate.
- Measurement of pro-factor D plasma concentrations in Danish blood donors.
Main Results:
- The developed ELISA demonstrated a working range of 0.82-25 ng/ml with acceptable CVs and >90% recovery.
- Median plasma concentration of pro-factor D in Danish blood donors was 134 ng/ml.
- Pro-factor D constitutes 8-15% of total factor D in circulation, and blood sampling significantly affects measured levels.
Conclusions:
- The study successfully established a validated ELISA for pro-factor D quantification.
- A significant proportion of factor D circulates as a pro-form, with levels influenced by sample handling.
- Further research is needed to understand the regulation and functional implications of pro-factor D and MASP-3 activation.
Abstract:
Factor D (also known as adipsin) is a serine protease and part of the complement system, involved in innate immune responses and effector functions of antibodies. Factor D cleaves factor B complexed with C3b, leading to the C3 convertase C3bBb. This C3 convertase is central in the alternative activation pathway and the amplification loop, which amplifies the two other complement activation pathways: the classical pathway and the lectin pathway. Adipocytes synthesize factor D as a pro-form comprising 6 additional residues that must be cleaved off to generate a mature form. The MBL-associated serine protease 3 (MASP-3), found in complex with the pattern recognition molecules of lectin activation pathway, converts the pro-form to mature factor D, which reportedly is the most abundant form found in the circulation at concentrations of 1-2 μg/ml among healthy individuals. The mature factor D is rate-limiting for complement activation, but little is known about the distribution of pro vs. mature factor D in the circulation, the regulation hereof and the potential activation stimuli of the lectin pathway, responsible for activation of MASP-3 and subsequent conversion of pro-form of factor D. In this light we established and validated an ELISA specific for measuring the pro-form of complement factor D. With a working range of 0.82-25 ng/ml, acceptable intra and inter assay CVs, and a relative recovery rate above 90%, we found that the median plasma concentration in Danish blood donors was 134 ng/ml; corresponding to that 8-15% factor D circulates as pro-form. We also found that blood sampling procedures affect conversion and hence the levels measured in serum and plasma.
