A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning and tissue localization.
A cysteine protease inhibitor with an apparent Mr = 12,600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08 nM. It also inhibits cathepsin L (Ki = 0.17 nM) and ficin (Ki = 0.52 nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.
Agarwala
K L
KL
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Kawabata
S
S
Hirata
M
M
Miyagi
M
M
Tsunasawa
S
S
Iwanaga
S
S
D000373:Agglutinins / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006434:Hemocytes / Q000737:chemistry* / Q000648:ultrastructure; D006737:Horseshoe Crabs / Q000737:chemistry* / Q000166:cytology; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry
D000373:Agglutinins; D000900:Anti-Bacterial Agents; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D010206:Papain