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Nε(γ-glutamyl)lysine crosslinks in the blood clot of the horseshoe crab, Limulus polyphemus
Vol. 188, No. 2, 1992 October 30, 1992
BIOCHEMICAL
AND BIOPHYSICAL
I@(-pGLUTAMYL)LYSINB BLOOD
CLOT
James Wilsonl,
IDepartment
OF
CROSSLINKS
THE HORSESHOE CRAB,
Frederick
RESEARCH COMMUNICATIONS Pages 655-661
IN TEE
LIMULUS
R. Rickles2t3, Peter and Laszlo Lorandlr2
POLYPHEMUS
B. Armstrong2r4rr
of Biochemistry, Molecular Biology and Cell Northwestern University, Evanston, IL 60208
Biology,
2Marine Biological Laboratory, Woods Hole, MA 02543 3Division of Hematology/Oncology, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06043 3veterans Administration Medical Center, Newington, CT 06111 4Laboratory for Cell Biology, Department of Zoology, University of California, Davis, CA 95616-8755 Received
September
1,
1992
Clots were allowed to form in samples of whole blood taken from the Amreican horseshoe crab, Limulus nolvohemus, in the absence and presence of dansylcadaverine (16), and were analyzed for their contents of NE(y-glutamyl)lysine and yglutamyl-dansylcadaverine. Clots obtained without dansylcadaverine yielded significant amounts of NE(rClots formed in the presence of glutamyl)lysine product. dansyldacaverine yielded only y-glutamyl-dansylcadaverine. Formation of these products reflects on the activity of transglutaminase released from the blood cells during 0 1992Academic mess, Inc. coagulation.
SUMMARY:
Blood is initiated coagulogen
coagulation in the horseshoe crab Limulus nolvnhemus by the proteolysis of the clottable protein, (12,14,19). Thereupon the cleaved monomer, coagulin,
*TO whom correspondence should be addressed at Department of Zoology, University Cell Biology, California, Davis, CA 95616-8755.
Laboratory of
Abbreviations: DC, dansylcadaverine (N-(5-aminopentyl)-5(dimethylamino)-1-naphthalenesulfonamide); EDTA, ethylenediaminetetraacetic acid; y-GACT, y-glutamylamine cyclotransferase; y-GluDc, y-glutamyldansylcadaverine; [2-hydroxyethyllpiperazine-N'-[2-ethanesulfonic acid]: performance liquid chromatography; TCA, trichloroacetic TEA, triethylamine.
for
HEPES, NHPLC, high acid; 0006-291X/92
655
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
polymerizes into the fibrillar structure of the clot network The zymogens of the participating proteases, as well as coagulogen itself are contained within the cytoplasmic vesicles of the amebocyte, the sole blood cell in the general circulation of Limulus (17). All of these constituents are released from the cells by exocytosis (1,3,4,13). The extracellular clot is presumed to have the functions of arresting bleeding as well as entrapping and immobilizing bacteria that would otherwise disseminate beyond the wound site and gain access to the hemocoel (4,13). Tissue transglutaminase released from cells has been shown to play an important role in the clotting of the plasma of some invertebrates (10,15); furthermore in 1973, Campbell-Wilkes discovered a potent Ca++-dependent transglutaminase in Limuu polvohemus amebocytes (5), a finding later confirmed by Chung et al. (8). The enzyme has recently been purified (21). In order to evaluate the participation of transglutaminase in the we undertook a direct search for coagulation of blood in Limulus, the presence of NE(v-glutamyl)lysine crosslinks in clots produced by cultured amebocytes. (19)
l
MATERIALS
AND METHODS
The coaculin
clot
Blood was obtained from adult specimens of Limulus golvnhemus under sterile, endotoxin-free conditions by cardiac puncture (2). Six drops of blood were collected into 100 mm diameter bacteriologic polystyrene Petri dishes (Fisher) containing 10 ml of sterile, endotoxin-free 3% NaCl solution (Travenol Laboratories,Deerfield, IL.: a 1:50 dilution of the blood) with or without 0.5 mM DC (Sigma, St Louis, MO). The final concentration of Ca++ was estimated to be 0.2 mM. Under these conditions, the blood cells attached and flattened on the surface of the dish and degranulated over a period of lo-30 minutes. The clots that developed in association with the monolayers of Limulus amebocytes cultured in the Dc-containing medium appeared to be similar to those associated with cells in DC-free medium. The control and DC-containing clots were removed from the dish with a rubber policeman and were frozen in 3% NaCl until analysis. HPLC analysis
of
NE-Iv-olutamvl)lvsine
The control clot and the one produced in the presence of 0.5 mM DC were thawed to room temperature and were centrifuged to separate the clot liquor from the insoluble clot matrix. The pellets of clot matrices were washed with 1 ml of Ca+2 and Mg+2free sea water, pH 7.0 (460 mM NaCl; 7 mM Na2SO4: 10 mM KCl; 2.5 mM EDTA; 10 mM HEPES buffer, pH 7.0) and the washing solutions were combined with the clot liquor. TCA was added both to the clot liquor and clot matrix fractions to 10% final concentration. 656
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The TCA-insoluble material in each sample was washed three times with 1 ml of 5% TCA, twice with 1:l ethanol:acetone, twice with acetone, and was dried under vacuum (Speed Vat Concentrator: Savant Instruments, Hicksville, NY). The samples were incubated for 1 hr in 1 ml of 0.1 M NaOH, which dissolved all of the material precipitated by TCA from the clot liquor but only part of the TCA-extracted pellets of clot matrices. Ten ~1 of 5 M Hcl was added and the samples were dialyzed exhaustively against 0.1 M NH4HC03 (Spectropor 3 dialysis membrane, Baxter Scientific). Following dialysis the samples were subjected to enzymatic digestion by the sequential addition of proteases utilizing the following enzymes (6): subtilisin (2 X 20 pg, Sigma Type VIII); pronase (2 X 20 pg, Calbiochem, La Jolla, CA): carboxypeptidase Y leucine aminopeptidase (2 X 7.5 pg, Sigma Type (20 pg, Sigma): III-CP); and prolidase (7.5 I-(g, Sigma). At the conclusion of enzymatic hydrolysis, the samples were dried, redissolved in water, and dried again. The samples were finally dissolved in water, centrifuged, and the supernatant subjected to analysis. Amino acid analysis was undertaken in order to determine the amount of enzymatically digested protein in each sample. Aliguots (3 ~1) were dried and redissolved in 10 1.r1 water and 50 ~1 of 20% (v/v) TEA in ethanol was added (20). The materials were dried and the above treatment was repeated twice more prior to dissolving the samples in water for analysis. Amino acid standards (Amino Acid Standard H, Pierce Chemical Co., Rockford, IL) containing glutamine, asparagine and tryptophan (Sigma) were processed in the same manner. Amino acid analysis using precolumn derivatization with ortho-phtalaldehyde was performed according to Griffin et al. (11). The amount of free amino acids in a sample was totaled, and the amount of free amino acids found in the enzyme control was subtracted to estimate the amount of digested protein substrate. The enzyme control contained all of the proteases, but not the clot matrix or liquor. Estimation of NE(y-glutamyl)lysine was performed by reverse phase HPLC with a Zorbax C9 COlUmn (DuPont, Wilmington, DE) Using the Waters Associates HPLC apparatus as previously described (6). An aliguot of each sample containing a known amount of digested protein, either with or without the addition of a known amount of an NE(y-glutamyl)lysine standard, was dried, redissolved in 10 ~1 water and 100 ~1 20% TEA in ethanol and then redried. The sample was redissolved in the same solvent, redried, and redissolved in 25 ~1 50 mM sodium phosphate, pH 7.5, either with 5 ~1 of 5 mN sodium phosphate pH 7.5 or 5 1.c1 y-GACT in the sodium phosphate buffer [O.l activity units/ml vs. N&(y-glutamyl)lysine] (9). The samples were incubated at 37OC, 30 min. and then 10 ~1 of 0.3 M HCl was added. Pre-column derivatization with o-phthaldehyde on 10 ~1 of samples was performed using the WISP as previously described (6). The gradient for separating NE(y-glutamyl)lysine was altered slightly from previous reports (6) as follows: (1) the pH of the 40 mM potassium acetate buffer was increased to pH 6.4; (2) after the methanol concentration reached 36% at 6.6 min., it was increased linearly to reach 54% at 23.5 min. with no Chemical synthesis of y-GluDc and reverseflow rate change. phase HPLC separation of y-GluDc and DC were performed as described elsewhere (7). Treatment of DC-labeled samples with yGACT was similar to the above procedure except that no drying 30 ~1 of DC-treated clot matrix or clot with TEA was required: liquor was mixed with 45 ~1 of 100 mN sodium phosphate buffer, pH 7.5 then 10 ~1 of 10 mM HCl or 10 ~1 of a solution containing 657
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.01 mM y-GluDc and 0.01 mM DC in 10 mM HCl was added, followed by addition of 5 ~1 of 5mM sodium phosphate or 5 ~1 of y-GACT as The samples were incubated at 37O C for 1 h, described above. then 10 ~1 of 0.6 M HCl was added and 80 ~1 of the sample was injected into the HPLC column. Molar frequencies of the isolated NE(y-glutamyl)lysine and yGluDc products are given for 100,000 g of protein. RESULTS AND DISCUSSION Following o-phtalaldehyde derivatization of the peptide digest of Limulus clots obtained in the absence of DC, peaks of NE(y-glutamyl)lysine emerged between 16.83 and 16.95 minutes from Authenticity of the peak was the analytical HPLC column. established through augmentation with known amounts of fl(yTreatment of the digest by y-GACT, an glutamyl)lysine peptide. enzyme capable of cleaving the NE(y-glutamyl)lysyl bond, still left a residual peak at this location. To reliably estimate the amount of NE(y-glutamy)lysine recovered from the experimental samples, the residual peak area was subtracted in the calculations. The digests of the clots prepared in the presence of DC showed a complete lack of NE(y-glutamyl)lysine but revealed instead the characteristic y-GluDc adduct (emerging at 10.2 min), which was monitored directly by flurorescence (7). The authenticity of this peak was verified by augmentation with known quantities of y-GluDc as well as by cleavage of y-GluDc with yGACT, which in this case gave rise to the later-emerging (11.75 min) fluorescent DC product (Fig. 1). Table 1 summarizes our measurements for the frequencies of @((y-glutamyl)lysine cross links in the clot obtained without DC and for the y-GluDc adduct isolated from the clot obtained in the presence of DC. The data clearly reflect the presence of significant amounts of NE(y-glutamyl)lysine crosslinks (0.3 moles/l05 g.of protein) in the matrix of the Limulus clot collected in the absence of DC. When this alternate substrate of transglutaminase was included in the clotting mixture, the above crosslinks were replaced in the clot matrix by blocked y-GluDc with essentially the same frequency (0.28 moles/105 g. of protein). The measured values are thought to underestimate the true frequencies of tissue transglutaminase products because of incomplete solubility of the initial TCA precipitate of the clot matrix and the possibility of incomplete digestion of the clot structure during the subsequent steps of proteolysis.
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A
FIG. 1. HPLC profile of y-GluDc isolated from the matrix of a clot formed in the presence of DC. Panel A shows the fluorescence of the product derived from 17.8 pg of the digest of the clot matrix: the main peak emerging at 10.20 min corresponds to r-GluDc and the small peak (11.75 min) represents a tiny amount of residual DC. Panel B is the material shown in Panel A after treatment of the sample with yGACT; note the disappearance of y-GluDc and the formation of Dc. Panel C is the sample shown in Panel A with the addition of 80 p moles of y-GluDc and DC. Panel D shows the material analyzed in Panel C, but after treatment with I-GACT; again this enzyme eliminated all yGluDc, producing free Dc.
In summary, our experiments show that transglutaminasedependent crosslinking occurs during clot formation in Lm blood. This contrasts with the conclusion of a previous report that such crosslinks do not exist (18). In that study, blood cells were lysed with distilled water and the cell-free extract was induced to clot by the addition of bacterial
TABLE 1: Frequencies (in moles/l05 NE(y-glutamyl)lysine and y-GluDc isolated Clotting conditions Without With
DC
Sample
Dc
Clot Clot Clot Clot
g. of protein) of from the Limulus clot
NE(y-glutamyl)lysine
matrix liquor matrix liquor
0.3 0.07 0.0 0.0 659
y-GluDc
0 0 0.28 0.06
Vol.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In the present study, clots were allowed to lipopolysaccharide. It is possible that form in the proximity of living blood cells. a cell-associated transglutaminase was lost or inactivated during the extraction of the blood cells by the procedures used by Roth but was active during clotting when cells were present in et al., In this case, NE(y-glutamyl)lysine crosslinks would the system. be expected in the clot only in the latter situation. ACKNOWLEDGMENTS This research was supported by a USPHS Research 03512) and by grants from the National Institutes 38185 and HL-02212).
Career Award (HLof Health (GM
RBFEREMCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Armstrong, P.B. (1985a) in Blood Cells of Marine Invertebrates (Cohen, W., ed.) pp. 77-128, Alan R. Liss, New York. Armstrong, P.B. (1985b) in Blood Cells ef Marine Invertebrates (Cohen, W.D., ed.) pp. 251-279, Alan R. Liss, New York. Armstrong, P.B., and Rickles, F.R. (1982) Exp. Cell Res. 140, 15-24. Bang, F.B. (1979) Prog. Clin. Biol. Res. 29, 109-123. Campbell-Wilkes, L. (1973) Ph.D. Dissertation, Northwestern University. Univ. Microfilms, 73-30 763, Ann Arbor MI. Cariello, L, Wilson, J. and Lorand, L. (1984) Biochemistry 23, 6843-6850. Cariello, L., Velasco, P.T., Wilson, J., Parameswaran, K.N., Darush, F., and Lorand, L. (1990) Biochemistry 29, 5103-5108. Chung, S.I., Seid, R.C., and Liu, T.-Y. (1977) Thromb. Haemostas. 38, 182. Fink, ML., Chung, S.I., and Folk, J.E. (1980) Proc. Natl. Acad. Sci. USA. 77, 4564-4568. Fuller, G.M., and Doolittle, R.F. (1971) Biochemistry 10, 1311-1315. Griffin, M., Price, S.J., and Palmer, T. (1982) Clin. Chim. Acta 125, 89-95. Harada, T., Morita, T., Iwanaga, S., Nakamura, S., and Niwa, M. (1979) Prog. Clin. Biol. Res. 29, 209-220. Levin, J. and Bang, F.B. (1964) Bull. Johns Hopkins Hosp. 115, 337-345. Liu, T.-Y., Seid, R.S., Tai, J.Y., Liang, S.-M., Sakmar., T.P., and Robbins, J.B. (1979) Prog. Clin. Biol. Res. 29, 147-158. Lorand, L., Doolittle, R.F., Konishi, K., and Riggs, S.K. (1963) Arch. Biochem. Biophys. 102, 171-179. Lorand, L., Rule, N.G., Ong, H.H., Furlanetto, R., Jacobsen, A Downey, J., Oner, N., and Brunner-lorand, J. (1968) Bi&hemistry 7, 1214-1223. Miirer, E.H., Levin, J., and Holme, R. (1975) J. Cellul. Physiol. 86, 533-542. 660
Vol.
188, No. 2, 1992
18. 19. 20. 21. 22.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Roth, R.I., Chen, J.C.R., and Levin, J. (1989) Thromb. Res. SS, 25-36. Takagi, T., Hokama, Y., Morita, T., Iwanaga, S., Nakamura, S and Niwa, M. (1979) Prog. Clin. Biol. Res. 29, 169-184. T&r, G.E. (1982) In: Methods in Protein Seouence Analvsis (Elzinga, M., Ed.), pp. 223-232, Humana Press, Clifton, NJ. Tokunaga, F.,Yamada, M., Muta, T., Hiranaga-Kawabota, M., Iwanaga, S., Ichinose, A., and Davie, E.W. (1991) Thromb. Haemostas. 65, 936. Young, M.S., Levin, J., and Prendergast, R.A. (1972) J. Clin. Invest. 51, 1790-1797.
661
BIOCHEMICAL
AND BIOPHYSICAL
I@(-pGLUTAMYL)LYSINB BLOOD
CLOT
James Wilsonl,
IDepartment
OF
CROSSLINKS
THE HORSESHOE CRAB,
Frederick
RESEARCH COMMUNICATIONS Pages 655-661
IN TEE
LIMULUS
R. Rickles2t3, Peter and Laszlo Lorandlr2
POLYPHEMUS
B. Armstrong2r4rr
of Biochemistry, Molecular Biology and Cell Northwestern University, Evanston, IL 60208
Biology,
2Marine Biological Laboratory, Woods Hole, MA 02543 3Division of Hematology/Oncology, Department of Medicine, University of Connecticut Health Center, Farmington, CT 06043 3veterans Administration Medical Center, Newington, CT 06111 4Laboratory for Cell Biology, Department of Zoology, University of California, Davis, CA 95616-8755 Received
September
1,
1992
Clots were allowed to form in samples of whole blood taken from the Amreican horseshoe crab, Limulus nolvohemus, in the absence and presence of dansylcadaverine (16), and were analyzed for their contents of NE(y-glutamyl)lysine and yglutamyl-dansylcadaverine. Clots obtained without dansylcadaverine yielded significant amounts of NE(rClots formed in the presence of glutamyl)lysine product. dansyldacaverine yielded only y-glutamyl-dansylcadaverine. Formation of these products reflects on the activity of transglutaminase released from the blood cells during 0 1992Academic mess, Inc. coagulation.
SUMMARY:
Blood is initiated coagulogen
coagulation in the horseshoe crab Limulus nolvnhemus by the proteolysis of the clottable protein, (12,14,19). Thereupon the cleaved monomer, coagulin,
*TO whom correspondence should be addressed at Department of Zoology, University Cell Biology, California, Davis, CA 95616-8755.
Laboratory of
Abbreviations: DC, dansylcadaverine (N-(5-aminopentyl)-5(dimethylamino)-1-naphthalenesulfonamide); EDTA, ethylenediaminetetraacetic acid; y-GACT, y-glutamylamine cyclotransferase; y-GluDc, y-glutamyldansylcadaverine; [2-hydroxyethyllpiperazine-N'-[2-ethanesulfonic acid]: performance liquid chromatography; TCA, trichloroacetic TEA, triethylamine.
for
HEPES, NHPLC, high acid; 0006-291X/92
655
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
polymerizes into the fibrillar structure of the clot network The zymogens of the participating proteases, as well as coagulogen itself are contained within the cytoplasmic vesicles of the amebocyte, the sole blood cell in the general circulation of Limulus (17). All of these constituents are released from the cells by exocytosis (1,3,4,13). The extracellular clot is presumed to have the functions of arresting bleeding as well as entrapping and immobilizing bacteria that would otherwise disseminate beyond the wound site and gain access to the hemocoel (4,13). Tissue transglutaminase released from cells has been shown to play an important role in the clotting of the plasma of some invertebrates (10,15); furthermore in 1973, Campbell-Wilkes discovered a potent Ca++-dependent transglutaminase in Limuu polvohemus amebocytes (5), a finding later confirmed by Chung et al. (8). The enzyme has recently been purified (21). In order to evaluate the participation of transglutaminase in the we undertook a direct search for coagulation of blood in Limulus, the presence of NE(v-glutamyl)lysine crosslinks in clots produced by cultured amebocytes. (19)
l
MATERIALS
AND METHODS
The coaculin
clot
Blood was obtained from adult specimens of Limulus golvnhemus under sterile, endotoxin-free conditions by cardiac puncture (2). Six drops of blood were collected into 100 mm diameter bacteriologic polystyrene Petri dishes (Fisher) containing 10 ml of sterile, endotoxin-free 3% NaCl solution (Travenol Laboratories,Deerfield, IL.: a 1:50 dilution of the blood) with or without 0.5 mM DC (Sigma, St Louis, MO). The final concentration of Ca++ was estimated to be 0.2 mM. Under these conditions, the blood cells attached and flattened on the surface of the dish and degranulated over a period of lo-30 minutes. The clots that developed in association with the monolayers of Limulus amebocytes cultured in the Dc-containing medium appeared to be similar to those associated with cells in DC-free medium. The control and DC-containing clots were removed from the dish with a rubber policeman and were frozen in 3% NaCl until analysis. HPLC analysis
of
NE-Iv-olutamvl)lvsine
The control clot and the one produced in the presence of 0.5 mM DC were thawed to room temperature and were centrifuged to separate the clot liquor from the insoluble clot matrix. The pellets of clot matrices were washed with 1 ml of Ca+2 and Mg+2free sea water, pH 7.0 (460 mM NaCl; 7 mM Na2SO4: 10 mM KCl; 2.5 mM EDTA; 10 mM HEPES buffer, pH 7.0) and the washing solutions were combined with the clot liquor. TCA was added both to the clot liquor and clot matrix fractions to 10% final concentration. 656
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The TCA-insoluble material in each sample was washed three times with 1 ml of 5% TCA, twice with 1:l ethanol:acetone, twice with acetone, and was dried under vacuum (Speed Vat Concentrator: Savant Instruments, Hicksville, NY). The samples were incubated for 1 hr in 1 ml of 0.1 M NaOH, which dissolved all of the material precipitated by TCA from the clot liquor but only part of the TCA-extracted pellets of clot matrices. Ten ~1 of 5 M Hcl was added and the samples were dialyzed exhaustively against 0.1 M NH4HC03 (Spectropor 3 dialysis membrane, Baxter Scientific). Following dialysis the samples were subjected to enzymatic digestion by the sequential addition of proteases utilizing the following enzymes (6): subtilisin (2 X 20 pg, Sigma Type VIII); pronase (2 X 20 pg, Calbiochem, La Jolla, CA): carboxypeptidase Y leucine aminopeptidase (2 X 7.5 pg, Sigma Type (20 pg, Sigma): III-CP); and prolidase (7.5 I-(g, Sigma). At the conclusion of enzymatic hydrolysis, the samples were dried, redissolved in water, and dried again. The samples were finally dissolved in water, centrifuged, and the supernatant subjected to analysis. Amino acid analysis was undertaken in order to determine the amount of enzymatically digested protein in each sample. Aliguots (3 ~1) were dried and redissolved in 10 1.r1 water and 50 ~1 of 20% (v/v) TEA in ethanol was added (20). The materials were dried and the above treatment was repeated twice more prior to dissolving the samples in water for analysis. Amino acid standards (Amino Acid Standard H, Pierce Chemical Co., Rockford, IL) containing glutamine, asparagine and tryptophan (Sigma) were processed in the same manner. Amino acid analysis using precolumn derivatization with ortho-phtalaldehyde was performed according to Griffin et al. (11). The amount of free amino acids in a sample was totaled, and the amount of free amino acids found in the enzyme control was subtracted to estimate the amount of digested protein substrate. The enzyme control contained all of the proteases, but not the clot matrix or liquor. Estimation of NE(y-glutamyl)lysine was performed by reverse phase HPLC with a Zorbax C9 COlUmn (DuPont, Wilmington, DE) Using the Waters Associates HPLC apparatus as previously described (6). An aliguot of each sample containing a known amount of digested protein, either with or without the addition of a known amount of an NE(y-glutamyl)lysine standard, was dried, redissolved in 10 ~1 water and 100 ~1 20% TEA in ethanol and then redried. The sample was redissolved in the same solvent, redried, and redissolved in 25 ~1 50 mM sodium phosphate, pH 7.5, either with 5 ~1 of 5 mN sodium phosphate pH 7.5 or 5 1.c1 y-GACT in the sodium phosphate buffer [O.l activity units/ml vs. N&(y-glutamyl)lysine] (9). The samples were incubated at 37OC, 30 min. and then 10 ~1 of 0.3 M HCl was added. Pre-column derivatization with o-phthaldehyde on 10 ~1 of samples was performed using the WISP as previously described (6). The gradient for separating NE(y-glutamyl)lysine was altered slightly from previous reports (6) as follows: (1) the pH of the 40 mM potassium acetate buffer was increased to pH 6.4; (2) after the methanol concentration reached 36% at 6.6 min., it was increased linearly to reach 54% at 23.5 min. with no Chemical synthesis of y-GluDc and reverseflow rate change. phase HPLC separation of y-GluDc and DC were performed as described elsewhere (7). Treatment of DC-labeled samples with yGACT was similar to the above procedure except that no drying 30 ~1 of DC-treated clot matrix or clot with TEA was required: liquor was mixed with 45 ~1 of 100 mN sodium phosphate buffer, pH 7.5 then 10 ~1 of 10 mM HCl or 10 ~1 of a solution containing 657
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
0.01 mM y-GluDc and 0.01 mM DC in 10 mM HCl was added, followed by addition of 5 ~1 of 5mM sodium phosphate or 5 ~1 of y-GACT as The samples were incubated at 37O C for 1 h, described above. then 10 ~1 of 0.6 M HCl was added and 80 ~1 of the sample was injected into the HPLC column. Molar frequencies of the isolated NE(y-glutamyl)lysine and yGluDc products are given for 100,000 g of protein. RESULTS AND DISCUSSION Following o-phtalaldehyde derivatization of the peptide digest of Limulus clots obtained in the absence of DC, peaks of NE(y-glutamyl)lysine emerged between 16.83 and 16.95 minutes from Authenticity of the peak was the analytical HPLC column. established through augmentation with known amounts of fl(yTreatment of the digest by y-GACT, an glutamyl)lysine peptide. enzyme capable of cleaving the NE(y-glutamyl)lysyl bond, still left a residual peak at this location. To reliably estimate the amount of NE(y-glutamy)lysine recovered from the experimental samples, the residual peak area was subtracted in the calculations. The digests of the clots prepared in the presence of DC showed a complete lack of NE(y-glutamyl)lysine but revealed instead the characteristic y-GluDc adduct (emerging at 10.2 min), which was monitored directly by flurorescence (7). The authenticity of this peak was verified by augmentation with known quantities of y-GluDc as well as by cleavage of y-GluDc with yGACT, which in this case gave rise to the later-emerging (11.75 min) fluorescent DC product (Fig. 1). Table 1 summarizes our measurements for the frequencies of @((y-glutamyl)lysine cross links in the clot obtained without DC and for the y-GluDc adduct isolated from the clot obtained in the presence of DC. The data clearly reflect the presence of significant amounts of NE(y-glutamyl)lysine crosslinks (0.3 moles/l05 g.of protein) in the matrix of the Limulus clot collected in the absence of DC. When this alternate substrate of transglutaminase was included in the clotting mixture, the above crosslinks were replaced in the clot matrix by blocked y-GluDc with essentially the same frequency (0.28 moles/105 g. of protein). The measured values are thought to underestimate the true frequencies of tissue transglutaminase products because of incomplete solubility of the initial TCA precipitate of the clot matrix and the possibility of incomplete digestion of the clot structure during the subsequent steps of proteolysis.
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A
FIG. 1. HPLC profile of y-GluDc isolated from the matrix of a clot formed in the presence of DC. Panel A shows the fluorescence of the product derived from 17.8 pg of the digest of the clot matrix: the main peak emerging at 10.20 min corresponds to r-GluDc and the small peak (11.75 min) represents a tiny amount of residual DC. Panel B is the material shown in Panel A after treatment of the sample with yGACT; note the disappearance of y-GluDc and the formation of Dc. Panel C is the sample shown in Panel A with the addition of 80 p moles of y-GluDc and DC. Panel D shows the material analyzed in Panel C, but after treatment with I-GACT; again this enzyme eliminated all yGluDc, producing free Dc.
In summary, our experiments show that transglutaminasedependent crosslinking occurs during clot formation in Lm blood. This contrasts with the conclusion of a previous report that such crosslinks do not exist (18). In that study, blood cells were lysed with distilled water and the cell-free extract was induced to clot by the addition of bacterial
TABLE 1: Frequencies (in moles/l05 NE(y-glutamyl)lysine and y-GluDc isolated Clotting conditions Without With
DC
Sample
Dc
Clot Clot Clot Clot
g. of protein) of from the Limulus clot
NE(y-glutamyl)lysine
matrix liquor matrix liquor
0.3 0.07 0.0 0.0 659
y-GluDc
0 0 0.28 0.06
Vol.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
In the present study, clots were allowed to lipopolysaccharide. It is possible that form in the proximity of living blood cells. a cell-associated transglutaminase was lost or inactivated during the extraction of the blood cells by the procedures used by Roth but was active during clotting when cells were present in et al., In this case, NE(y-glutamyl)lysine crosslinks would the system. be expected in the clot only in the latter situation. ACKNOWLEDGMENTS This research was supported by a USPHS Research 03512) and by grants from the National Institutes 38185 and HL-02212).
Career Award (HLof Health (GM
RBFEREMCES 1.
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