Separation of large and small granules from horseshoe crab (Tachypleus tridentatus) hemocytes and characterization of their components.
We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.
Shigenaga
T
T
Department of Molecular Biology, Graduate School of Medical Science, Fukuoka.
Takayenoki
Y
Y
Kawasaki
S
S
Seki
N
N
Muta
T
T
Toh
Y
Y
Ito
A
A
Iwanaga
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D001798:Blood Proteins / Q000302:isolation & purification*; D002457:Cell Extracts / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006434:Hemocytes / Q000648:ultrastructure*; D006737:Horseshoe Crabs / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000648:ultrastructure
D000890:Anti-Infective Agents; D001798:Blood Proteins; D002457:Cell Extracts; D010455:Peptides