Structure and bioactivity of recombinant human CTAP-III and NAP-2

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are derived from platelet basic protein (PBP).
• These peptides are released from activated platelets and play roles in inflammation and wound healing.
• Understanding their distinct biological activities and structural properties is crucial.

Purpose of the Study:

• To clone and express recombinant CTAP-III and NAP-2 in E. coli.
• To characterize the biological activities and structural properties of these recombinant peptides.
• To elucidate the molecular basis for their differing functions.

Main Methods:

• Cloning of PBP cDNA and expression of CTAP-III and NAP-2 in E. coli.
• Purification and renaturation of recombinant proteins.
• In vitro bioassays including histamine release, neutrophil chemotaxis, shape change, IL-8 displacement, disulfide bond identification, and oligomerization studies (gel permeation chromatography, SDS/PAGE).

Main Results:

• Recombinant CTAP-III and NAP-2 were successfully produced and characterized.
• CTAP-III induced significant histamine release from basophils, while NAP-2 showed minimal activity.
• NAP-2 demonstrated potent chemotactic activity, shape change induction, and IL-8 displacement in neutrophils; CTAP-III lacked these activities.
• Disulfide bonds were identified in predicted positions.
• CTAP-III predominantly formed tetramers and dimers, whereas NAP-2 existed mainly as dimers at physiological concentrations.

Conclusions:

• Recombinant CTAP-III and NAP-2 exhibit distinct biological functions.
• NAP-2 is a potent neutrophil activator, while CTAP-III primarily affects basophils.
• Oligomerization, particularly tetramer formation of CTAP-III, may influence its biological activity.