Abstract
The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.
Highlights
Heparin is a highly sulfated glycosaminoglycan (GAG)3 made up of a mixture of polymers with a similar backbone of repeating hexuronic acid (␣-L-iduronic acid or -D-glucuronic acid) linked 1,4 to ␣-D-glucosamine units
Our results reveal the occurrence of two sulfated glycans in the hemolymph of this ascidian: heparin, which is present in intracellular granules of a circulating basophil-like cell, and a sulfated galactoglucan, which occurs free in the plasma
These results show for the first time the presence of heparin in a circulating basophil-like cell in an invertebrate chordate and may contribute toward the understanding of the evolution of the immune system in this phylum
Summary
Heparan sulfate from human aorta was extracted and purified as described previously [36]. Chondroitin 4-sulfate from whale cartilage, dermatan sulfate and heparin from porcine intestinal mucosa (140 units/mg), twice-crystallized papain (15 units/mg protein), and the standard disaccharides ␣-⌬UA-134-GlcN(SO4), ␣-⌬UA-134-GlcNAc(6SO4), ␣-⌬UA(2SO4)-134-GlcNAc, ␣-⌬UA(2SO4)-134-GlcN(SO4), ␣-⌬UA(2SO4)-134-GlcNAc(6SO4), ␣-⌬UA-134GlcN(SO4)(6SO4), and ␣-⌬UA(2SO4)-134-GlcN(SO4)(6SO4) were purchased from Sigma; chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter aurescens, chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris, and heparan sulfate lyase (EC 4.2.2.8) and heparin lyase (EC 4.2.2.7) from Flavobacterium heparinum were from Seikagaku America Inc. Agarose (standard low Mr) was obtained from Bio-Rad; toluidine blue was from Fisher; 1,9-dimethylmethylene blue was from Serva Feinbiochimica (Heidelberg, Germany); human antithrombin and thrombin were from Hematologic Technologies Inc. or from Hyphen Biomed; and thrombin chromogenic substrate tosyl-Gly-Pro-Arg-p-nitroanilide acetate (Chromozyn TH) was from Roche Applied Science. Chloroform (Merck) was used fresh for the organic extraction procedure
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