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The cancer-associated antigens Ca 125 and Ca 19-9 were demonstrated by radioimmunoassay to form structural units of a mucus glycoprotein in human milk taken from healthy women four days after parturition. The glycoprotein precipitated with the casein fraction at pH 4.6 and was completely absent in the whey as judged from Ca 19-9 assay. It could be effectively enriched by phenol-saline extraction from soluble milk proteins and further purified by gel filtration on Sephacryl S300 and Sephacryl S400.The active component with a bouyant density of 1.41 g/ml in isopycnic density gradient centrifugation (CsCl) shared common physico-chemical and chemical characteristics of mucus glycoproteins. Carbohydrates representing about 68% by weight were conjugated to protein by alkali-labile linkages, exclusively and were essentially free of D-mannose.Activities of Ca 125 and Ca 19-9 were both destroyed by treatment with periodate, mild alkali or neuraminidase suggesting the antigens are sialylated saccharides bound to protein by alkali-labile linkages. The fraction of monosialylated saccharide alditols isolated after reductive p-elimination from the mucus glycoprotein was shown to inhibit monoclonal antibodies anti-(Ca 125) and anti-(Ca 19-9) in radioimmunoassay.

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The primary structures of four major sialylated saccharide alditols derived from mucus glycoproteins of human seminal plasma were established by fast-atom-bombardment mass spectrometry and methylation analysis. Anomeric configurations of the glycosidic bonds were determined by exoglycosidase digestion and C r 0 3 oxidation.GalN Ac-01 Based on quantitative data from high-pressure liquid chromatography and on structural information, the biosynthetic pathways for neutral and sialylated saccharides related to the Lewis system were proposed. Expression of saccharides A 6, A 7 and A 8 and their asialo counterparts, which are characterized by antigenic determinants H, Le" and Ley, respectively, is qualitatively and quantitatively dependent on the Lewis blood type of the respective donor and correlates with Ca 19-9 activity of its seminal plasma.

The monoclonal antib ody-defined, tumor-associated antigen Ca 19-9, chemically identical with the sialylated Lewisa-carbohydrate determinant of a monoganglioside and a mucin, was demonstrated by radioimmunoassay to be present in large amounts as component of fucose-rich sialoglycoproteins, which had been extracted from human seminal plasma of healthy donors. The carbohydrate antigen of these glycoproteins (m > 205 kDa and m 11 5 kDa), which are presumably secreted by the prostatic gland, was absent in seminal plasma from bloodgroup-Lewis-negative men.The Ca 19-9 active sialyl-oligosaccharide was cleaved from the proteins by mild alkaline borohydride treatment and was shown to chromatograph on gradient elution from DEAE-Sephadex with the fraction of monosialylated saccharide alditols (MS-SP). The asialo derivative of the major saccharide alditol in this fraction was composed The carbohydrate antigen, Ca 19-9, which is defined by a monoclonal antibody [l] and chemically is identical with sialyllacto-N-fucopentaose I1 [2] has been demonstrated in various studies to be associated with certain types of human malignancies i.e. gastrointestiwal and pancreatic carcinoma [3]. The glycolipid has also been detected in human meconium, but never in extracts of adult tissues derived from healthy persons indicating that it may be a tumor-associated embryonic antigen [2]. Contrasting with this, the antigen was demonstrable histochemically in the epithelium of normal mucosa of the gall bladder and endocervix and in some ductal epithelia of the pancreas and salivary gland by an indirect immunoperoxidase method [4]. Moreover, the unexpected occurrence of the carbohydrate antigen in normal human seminal plasma has been reported in a recent contribution [5]. From this preliminary investigation it became evident that the monoclonal antibody-defined Ca 19-9 antigen forms part of fucose-rich sialoglycoproteins, which, presumably, are secreted by the prostatic gland. We now report on the isolation and Enzyme. Neuraminidase (EC 3.2.1.18).structural characterization of the major carbohydrates on these glycoproteins by methylation analysis and mass spectrometry. MATERIALS AND METHODS Extraction of seminal plasma glycoproteinsSemen was obtained by masturbation from healthy donors. Samples were centrifuged and the seminal plasma was removed for analysis in radioimmunoassay or preparation of the glycoproteins. Phenol/saline extraction of human seminal plasma (100 ml) was performed as described earlier [6]. Also samples from patients with aspermia as a consequence of bilateral occlusion of the ampullae were collected separately. These samples contain pure prostatic gland secretion [7]. SDS-polyacrylumide gel electrophoresisSamples of 2 mg glycoprotein (native sialoglycoprotein fraction, neuraminidase-treated or lectin-treated extract) dissolved in 1 ml Tris/glycine buffer pH 8.9 containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol were heated at 100°C for 5 min. Slab gel electrophoresis was performed in 7.5% acrylamide gel, 0.1% SDS. Gels w...