Limulus intracellular coagulation inhibitor type 3. Purification, characterization, cDNA cloning, and tissue localization.
We reported that limulus intracellular coagulation inhibitor type-1 (LICI-1) (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J. Biol. Chem. 269, 542-547) and LICI type-2 (LICI-2) (Miura, Y., Kawabata, S. , Wakamiya, Y., Nakamura, T., and Iwanaga, S. (1995) J. Biol. Chem. 270, 558-565) found in the hemocyte lysate belong to the serpin family. The LICI-1 specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C (k1 = 2.5 x 10(6) M-1 s-1), whereas LICI-2 inhibits preferentially limulus clotting enzyme (k1 = 4.3 x 10(5) M-1 s-1). In our ongoing studies on limulus serpin, we found another inhibitor, named LICI type-3 (LICI-3), which strongly inhibits (1,3)-beta-D-glucan-sensitive serine protease, factor G (k1 = 3.9 x 10(5) M-1 s-1). Thus, the limulus hemolymph coagulation cascade is effectively regulated by at least the three endogenous serpins. LICI-3, newly identified in hemocytes, is a single chain glycoprotein with an apparent Mr = 53,000, the largest one among known limulus serpins. A cDNA sequence for LICI-3 coded a mature protein of 392 amino acids, of which 68 residues were confirmed by peptide sequencing. LICI-3 showed significant sequence similarity to LICI-1 (45.8% identity) and LICI-2 (33.7% identity). LICI-3 contained a putative reactive site, -Arg-Ser-, distinct from that of LICI-2 (-Lys-Ser-) but the same as that of LICI-1. Expression of LICI-3 mRNA was detected only in hemocytes, and not in heart, brain, stomach, intestine, coxal gland, and skeletal muscle. Immunoblotting of the hemocyte-derived large and small granules with antiserum against LICI-3 suggested that it is stored specifically in large granules, as in the case of LICI-1 and LICI-2, and is released in response to external stimuli.
Agarwala
K L
KL
Department of Molecular Biology, Graduate School of Medical Science, Faculty of Science, Kyushu University 33, Fukuoka 812-81, Japan.
Kawabata
S i
Si
Miura
Y
Y
Kuroki
Y
Y
Iwanaga
S
S
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins; D001483:Base Sequence; D001779:Blood Coagulation Factors / Q000037:antagonists & inhibitors; D001798:Blood Proteins / Q000737:chemistry; D002918:Chymotrypsin / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D023082:Defensins; D015870:Gene Expression; D005796:Genes; D006023:Glycoproteins / Q000235:genetics* / Q000502:physiology; D006737:Horseshoe Crabs / Q000235:genetics / Q000502:physiology*; D007447:Invertebrate Hormones / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000235:genetics* / Q000302:isolation & purification; D015843:Serpins; D014018:Tissue Distribution
D060829:Arthropod Proteins; D001779:Blood Coagulation Factors; D001798:Blood Proteins; D018076:DNA, Complementary; D023082:Defensins; D006023:Glycoproteins; D007447:Invertebrate Hormones; C101766:LICI-3 protein, Tachypleus tridentatus; D012333:RNA, Messenger; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D002918:Chymotrypsin