Limulus intracellular coagulation inhibitor type 3. Purification, characterization, cDNA cloning, and tissue...

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Horseshoe crab hemolymph coagulation is a critical immune defense mechanism.
• Limulus intracellular coagulation inhibitor type-1 (LICI-1) and type-2 (LICI-2) are known serpins regulating this cascade.
• The precise regulation of coagulation by endogenous inhibitors is crucial for immune response.

Purpose of the Study:

• To identify and characterize novel endogenous serpins involved in horseshoe crab hemolymph coagulation.
• To elucidate the inhibitory specificities and functional roles of newly discovered serpins.
• To understand the storage and release mechanisms of these inhibitors within hemocytes.

Main Methods:

• Serine protease inhibition assays to determine enzyme kinetics (k1 values).
• Peptide sequencing and cDNA analysis to determine protein structure and identify reactive sites.
• mRNA expression analysis using Northern blotting to identify tissue-specific expression.
• Immunoblotting to determine subcellular localization within hemocytes.

Main Results:

• A novel serpin, limulus intracellular coagulation inhibitor type-3 (LICI-3), was identified.
• LICI-3 strongly inhibits the (1,3)-beta-D-glucan-sensitive serine protease, factor G (k1 = 3.9 x 10^5 M^-1 s^-1).
• LICI-3 is a glycoprotein (Mr = 53,000) with sequence similarity to LICI-1 and LICI-2, containing an Arg-Ser reactive site.
• LICI-3 mRNA is exclusively expressed in hemocytes and stored in large granules, similar to LICI-1 and LICI-2.

Conclusions:

• The horseshoe crab hemolymph coagulation cascade is regulated by at least three endogenous serpins: LICI-1, LICI-2, and LICI-3.
• LICI-3 plays a significant role in inhibiting factor G, contributing to the precise regulation of coagulation.
• The specific storage and release of LICI-3 from hemocyte granules highlight its importance in the horseshoe crab innate immune response.