Horseshoe crab amebocyte lysate factor G subunit A

Details Horseshoe crab amebocyte lysate factor G subunit A Horseshoe crab amebocyte lysate factor G subunit A

1995-02-28

1998-08-18

Grimes, Eric

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

435226, C07K 14435, C07K 14745, C12N 950

Patent

active

057959625

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to a polypeptide derived from limulus (horseshoe crab) amebocytes shown by an amino acid sequence of (1.fwdarw.3)-.beta.-D-glucan sensitive factor (hereinafter abbreviated as factor G) and a DNA encoding thereof, particularly to a polypeptide shown by an amino acid sequence of subunit a of factor G and a DNA encoding thereof.


BACKGROUND OF THE INVENTION

Heretofore, determination of endotoxin with a limulus amebocyte lysate has been known. This method is based on coagulation of lysate with a very small amount of endotoxin, for example at about 10.sup.-9 g. It has been elucidated with the progress of biochemistry that this coagulation reaction consists of the stepwise activation mechanism of some coagulation factors. (Takanori Nakamura et al., Jpn. J. Bacteriol., 38, 781-803 (1983)). FIG. 1 shows the coagulation cascade reaction of Japanese horseshoe crab (Tachypleus tridentatus). The structures of three serine protease precursors (factor C, factor B and proclotting enzyme) and a clottable protein (coagulogen) in FIG. 1 have been elucidated by studies such as cDNA cloning (T. Muta et al. (1991) J. Biol. Chem., 265, 22426-22433 and 266, 6554-6561, and T. Miyata et al (1986) J. Biochem., 100, 213-220).
The lysate has been known to react with (1.fwdarw.3)-.beta.-D-glucan which exists in the cell walls of fungi and yeasts, at concentrations of 10.sup.-8 -10.sup.-9 g to trigger coagulation (see FIG. 1). Factor G responds to (1.fwdarw.3)-.beta.-D-glucans, initiating clot formation. It is reported that factor G is a serine protease precursor as well as the other factors and it is a glycoprotein composed of non-covalently associated subunits a (72 kDa) and b (37 kDa) (64th Annual Meeting of Japan Biochemical Society, 1991).
Additionally, subunit a of factor G has a specific binding site to (1.fwdarw.3)-.beta.-D-glucan and subunit b has serine protease region. Factor G is supposed to be activated and express serine protease activity by the binding of (1.fwdarw.3)-.beta.-D-glucan to subunit a. However, the complete structure of factor G remains unknown.


DISCLOSURE OF THE PRESENT INVENTION

The present invention was accomplished with these understandings and aims to isolate and purify the factor G derived from limulus amebocytes, particularly subunit a having a binding site with (1.fwdarw.3)-.beta.-D-glucan and to elucidate its complete structure.
Another object of the present invention is to provide a polypeptide having a (1.fwdarw.3)-.beta.-D-glucan binding site of a factor G and a DNA encoding thereof.
The other object of the present invention is to provide a polypeptide of subunit a of factor G and DNA encoding thereof.
Further object of the present invention is to provide a polypeptide of factor G containing subunit a and a DNA encoding thereof.
Therefore, the present invention relates to a single stranded DNA encoding for a polypeptide containing at least one motif structure shown by an amino acid sequence of (SEQ. ID No:34): Gln-Gln-Trp-Ser, a double stranded DNA composed of said DNA and its complementary DNA, and a polypeptide shown by said amino acid sequence.
Furthermore, the present invention relates to a single stranded DNA encoding for a polypeptide consisted of below mentioned amino acid sequence or a homologous sequence thereof, a double stranded DNA composed of said DNA and its complementary DNA and a polypeptide shown by said amino acid sequence (SEQ. ID No.2):


__________________________________________________________________________ Ser His Glu Pro Lys Trp Gln Leu Val Trp Ser Asp Glu Phe Thr Asn Gly Ile Ser Ser Asp Trp Glu Phe Glu Met Gly Asn Gly Leu Asn Gly Trp Gly Asn Asn Glu Leu Gln Tyr Tyr Arg Arg Glu Asn Ala Gln Val Glu Gly Gly Lys Leu Val Ile Thr Ala Lys Arg Glu Asp Tyr Asp Gly Phe Lys Tyr Thr Ser Ala Arg Leu Lys Thr Gln Phe Asp Lys Ser Trp Lys Tyr Gly Lys Ile Glu Ala Ly

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