Two major proteins from locust plasma are involved in coagulation and are specifically precipitated by laminarin, a beta-1,3-glucan.
Incubation of plasma of the locust Locusta migratoria, with laminarin induced the precipitation of two major proteins with molecular masses of about 260,000 (P260) and 85,000 Da (P85). This precipitation was not observed when other polysaccharides, such as curdlan, dextran, chitin, cellulose or mannan were used. P260 and P85 were purified to homogeneity by a single step on heparin-sepharose chromatography. Since all attempts to separate P260 from P85, other than the use of sodium dodecyl sulfate, were unsuccessful, it is likely that these two molecules form a complex non-covalently associated. Treatment of P260-P85 complex with N-glycosidase F showed that P260 did not appear to be glycosylated whereas 6% of P85 molecular mass was due to N-linked carbohydrates. On the other hand, no change in molecular masses of P260 or P85 was observed once the complex had been treated with lipase. SDS-PAGE and Western blots of plasma and serum stained with blue Coomassie for proteins or with highly specific polysera to P260 or P85, respectively, showed that P260 was only present in plasma and P85 remained in both samples. This indicates that P260 is likely to be one of the most abundant plasma proteins directly involved in the coagulation process in Locusta migratoria. The addition of plasma or P260-P85 complex to a hemocyte lysate supernatant prior to its activation by laminarin induced a lower protease as well as phenoloxidase activity compared with the control. This reduction of activities was not observed in the presence of serum or when P260-P85 complex was added to a fully activated proPO system.
Duvic
B
B
Laboratoire de Pathologie Comparée, URA INRA-CNRS 2209, Université de Montpellier II, France. duvic@crit.univ-montp2.fr
Brehélin
M
M
D000595:Amino Acid Sequence; D000818:Animals; D011232:Chemical Precipitation; D005936:Glucans / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006110:Grasshoppers / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D047071:beta-Glucans
D005936:Glucans; D019476:Insect Proteins; D011134:Polysaccharides; D047071:beta-Glucans; C008247:laminaran; C033363:beta-1,3-glucan; D006026:Glycoside Hydrolases