Mamoru Isemura

Characterization of fibronectin from fetal human plasma in comparison with adult plasma fibronectin

Fibronectin was purified from fetal human plasma and characterized in comparison with fibronectin from adult human plasma. Fetal plasma fibronectin had an amino-acid composition, immunological properties, and cell-attachment-promoting activity similar to those of adult plasma fibronectin. However, fetal plasma fibronectin was shown to have a distinct carbohydrate composition which is characterized by the presence of fucose. To ascertain the differences in the carbohydrate moiety, 14C-labeled glycopeptides were prepared and sequentially analyzed with columns of immobilized concanavalin A and lentil lectin. Glycopeptides from fetal plasma fibronectin contained a population of glycopeptides which bound to both lectin gels. Almost all of the glycopeptides in this population lost their ability to bind to lentil lectin upon fucosidase digestion, indicating that fetal plasma fibronectin possesses a substantial amount of fucosylated biantennary glycans. In contrast, glycopeptides from adult plasma fibronectin practically lacked such glycopeptides. Another difference observed was that fetal plasma fibronectin had a larger amount of concanavalin A-unbound glycopeptides than did adult plasma fibronectin. These results indicate the presence of age-related variation in glycosylation of human plasma fibronectin.

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.

A weaker gelatin-binding affinity and increased glycosylation of amniotic fluid fibronectin than plasma fibronectin

Human amniotic fluid fibronectin had different carbohydrate moieties from plasma fibronectin. Nearly 90% of glycopeptides released from amniotic fluid fibronectin was not bound by concanavalin A-Sepharose, whereas 75% of glycopeptides from plasma fibronectin was bound. Amniotic fluid fibronectin showed a significantly lower gelatin-binding affinity than plasma fibronectin at 25 degrees C. When the incubation temperature was lowered to 4 degrees C, no significant difference in this activity was found. Cell-attachment promoting activity of the two fibronectins was not significantly different.

Hormonal effects on the activities of glycosidases in the endometrium of rabbit uterus

The activities of several glycosidases in the lysosomal fraction of the uterine endometrium of rabbit were measured using 4-MU-glycosides as substrates. The specific activity of beta-N-acetylglucosaminidase was the highest, which was followed by beta-galactosidase, beta-glucuronidase, and alpha-galactosidase in this order. beta-Glucosidase had the lowest activity among the glycosidases examined. In order to examine the hormonal effects on these glycosidases, the lysosomal fractions were prepared from the uterine endometrium of the control, estrogen-treated, and progesterone-treated rabbits. In all glycosidases examined, except for beta-glucosidase, the specific activity was highest in the lysosome obtained from estrogen-treated rabbit. The specific activity in the lysosome from the progesterone-treated rabbit was between that from the estrogen-treated rabbit and that from control. Hormonal treatments, however, affected neither pH optimum curves nor isozyme patterns of these glycosidases.

Pattern of fibronectin distribution in human lung cancer.

SummaryThe pattern of fibronectin (FN) distribution in human lung cancer was studied by indirect immunofluorescent staining, and by the peroxidase-antiperoxidase method in a total of 60 surgical specimens. They comprised 8 small cell carcinomas, 4 large cell carcinomas, 19 squamous cell carcinomas, 28 adenocarcinomas, and 1 adenoid cystic carcinoma.Of the 60 specimens 13 were FN-positive. They included 4 large cell carcinomas, 4 small cell carcinomas, 3 poorly differentiated squamous cell carcinomas, and 2 poorly differentiated and 1 moderatety differentiated adenocarcinomas.On the other hand, none of the well differentiated carcinomas was FN-positive around tumor cells. Our data suggest that undifferentiated, or poorly differentiated carcinomas of the lung tend to be FN-positive.

Interaction of fibronectin with arginine—agarose

Fibronectin, a high-M, glycoprotein, interacts with many biological and biochemical substances (review [l-3]). The binding of fibronectin to gelatin allows its isolation by affinity chromatography [4]. Dissociation of fibronectin from gelatin-agarose by various amino compounds suggests the presence in fibronectin of specific binding sites for these compounds [5]. However, fibronectin has been reported to have very low affinity for arginine-agarose. For example, 0.1 M NaCl in 0.05M Tris-HCI buffer eluted the fibronectin bound to arginine-agarose in [6]. We have confirmed these results in a study on the interaction between porcine blood plasma fibronectin and arginine-agarose with a relatively low content of arginine [7]. We have found that arginine-agarose with a higher arginine content retains >90% of fibronectin even when it is applied in the presence of 0.5M NaCl. This paper describes the binding of porcine plasma fibronectin to arginine-agarose, which can be dissociated with certain amino compounds, and the localization of the arginine-binding domains in fibronectin.

Further characterization of porcine plasma fibronectin which contains fucosylated carbohydrate chains

Porcine plasma fibronectin and its functional four fragments produced by cathepsin B digestion were examined for biological, immunochemical and biochemical properties. Native fibronectin, 150-kDa and 130-kDa fragments exhibited similar cell attachment-promoting activity to each other. In an Ouchterlony double immunodiffusion system, these three polypeptides formed a precipitin line with anti-fibronectin antiserum, while the 50-kDa and 30-kDa fragments did not. The 150-kDa and 130-kDa fragments contained free sulfhydryl(s). The glycopeptide fractions were prepared by pronase digestion of porcine and human plasma fibronectin, and radiolabeled with [14C]acetic anhydride. The results of affinity chromatography with concanavalin A and lentil lectin immobilized on agarose indicated that the porcine glycopeptide fraction was different from the human fraction in that a larger part (58%) of the former was bound to lentil lectin. About 90% of this lentil lectin-reactive glycopeptides lost this reactivity upon alpha-L-fucosidase digestion. The glycopeptide fractions were also prepared from three carbohydrate-containing domains. Less than 30% of the radioactivity of the glycopeptide fractions of 150-kDa and 130-kDa fragments was retained on the lentil lectin-agarose, while about 90% of that from the 50-kDa fragment was retained. These results indicate that porcine plasma fibronectin has characteristics very similar to those of human plasma fibronectin and others, but is unique in that it contains fucosylated carbohydrate chains which unevenly distribute through functional domains.

Novel glycopeptides, containing desmosine and isodesmosine, isolated from porcine aorta

Intima-media of porcine thoracic aorta were digested with pronase, after extraction of the saline-soluble matters and fat. A glycopeptide fraction was precipitated with 90% (vol/vol) ethanol from the 80% ethanol-soluble fraction of the trichloroacetic acid (7%)-soluble fraction of the pronase digest. The glycopeptide fraction was fractionated by affinity chromatography on concanavalin A (Con A)-Sepharose 4B, yielding 4 fractions (FA, FB, FC and FD). The most carbohydrate-rich fraction (FB) was further purified to a homogeneous state. The purified FB (FB-0.1) and all other fractions contained desmosine and isodesmosine. The major sugars in the fractions without or with low affinity for Con A (FA, FB, and FB-0.1) were glucosamine, galactose, mannose and sialic acid, while those in the fractions with high affinity for this lectin (FC and FD) were glucosamine, glucose and mannose. All the fractions contained glycine, aspartic acid (and/or asparagine), serine, proline, threonine, glutamic acid (and/or glutamine) and alanine as the major amino acids, amounting to approximately 80% of the total.

Isolation and characterization of a blood group active glycopeptide from porcine aorta

A fucose-rich glycopeptide was prepared from the pronase digest of porcine thoracic aorta by gel-filtration through Sephadex G-100, DEAE-Sephadex A-25 column chromatography and alpha-amylase digestion. This glycopeptide was electrophoretically homogeneous. The large molecular size and chemical composition suggested that this glycopeptide was derived from mucin-type glycoprotein. The results of the beta-elimination reaction indicated that this glycopeptide contained the O-glycosidic linkages between galactosamine and serine/threonine. This glycopeptide exhibited blood group A and H activities. The present study revealed that the porcine thoracic aorta contains a blood group antigen of mucin-type glycoprotein nature.