Liver-intestine cadherin: molecular cloning and characterization of a novel Ca(2+)-dependent cell adhesion molecule expressed in liver and intestine.

A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI-cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.

UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation.

Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.

Decrease of human serum fucosyltransferase as an indicator of successful tumor therapy.

Surgical removal of colon carcinomas leads to a decrease in the rate of incorporation of [14C]fucose into its endogenous acceptor in human serum; normal incorporation rates are attained within 14 days. A similar time course has been determined for alpha2- and alpha3-fucosyltransferase when either desialo- or desialodegalactofetuin are employed as exogenous acceptors. A correlation has also been seen between transferase activity and the therapeutic response of patients with breast cancer. These results indicate that the determination of fucosyltransferase activity can facilitate the diagnosis of neoplasia, and the success of surgery, chemotherapy, or radiation.

Glycosyltransferases and glycosidases in Morris hepatomas.

The following three parameters were studied in Morris hepatomas of different growth rates: (a) the specific activity of guanosine dephosphate (GDP)-fucose:glycoprotein fucosyltransferase and cytidine monophosphate (CMP)-N-acetylneuraminic acid:glycoprotein sialyltransferase, (B) the content of GDP-fucosee and CMP-N-acetylneuraminic acid, and (c) the activity of alpha-L-fucosidase and neuraminidase. Fucosyltrasferase activities were significantly elevated in all hepatomas investigated. Especially high levels of enzyme were measured in the rapidly growing tumors 7777, 66, and 3924A. The increase varied between 2- and 3-fold when compared with the corresponding host liver. Conversely, the activity of the sialytransferase was greatly decreased in all hepatoma lines with a rapid or intermediate growth rate. In the fast-growing tumor 9618A2, the activity was reduced to 8%. GDP-fucose and CMP-N-acetylneuraminic acid were determined by the isotope dilution technique. In normal rat liver from Buffalo or ACl rats, the concentration of GDP-fucose was 6.5+/-0.9 and 9.5+/-1.1nmoles/g, wet weight, respectively. In the fast-growing hepatomas 3924A and 9121, levels up to 21.5 nmoles/g, wet weight, were found, However, the content of CMP-N-acetylneuraminic acid in hepatomas was indluenced to a lesser extent by the degree of differentiation of the tumor. In the most rapidly growing tumor, 9618A2, a level of alpha-L- fucosidase seven times higher than in host liver was determined. Moreover, there existed a correlation bewteen the age of the hepatoma and enzyme activity. Within the 2nd week after inoculation, fucosidase activity increased from 130 to 343 nmoles/hr/mg of protein. Neuraminidase was measured in a new linked assay system. The activity of this enzyme was lowered by 50% or was at least unchanged when compared to the activity in host liver. Our results indicate that specific alterations of fucose metabolism are a characteristic feature of Morris hepatomas.

Alterations of D-galactose metabolism in Morris hepatomas.

Studies on Morris hepatomas demonstrate that the specific activity of the enzymes of the Leloir pathway are subject to a variation in tumor tissue. The key enzyme of the galactose pathway, uridine diphosphogalactose 4'-epimerase, is elevated 5- to 9-fold in the rapidly growing and poorly differentiated Tumors 3924A and 7777; in line 9618A2, an even 28-fold increase was found. The observed correlation between enzyme activity, growth rate, and degree of differentiation of the hepatoma suggests that the differences are not coincidental variations. Conversely, the activity of uridine diphosphoglucose: galactose-1-phosphate uridyltransferase was diminished by 40 to 70% as compared to host liver, and the level of galactokinase showed only minor changes. The uptake of galactose and galactosamine by the hepatoma is heavily impaired, whereas the transport of other hexoses and amino sugars (2-deoxyglucose, L-fucose, N-acetylglucosamine, N-acetylmannosamine) is hardly affected. It appears that part of the carrier-mediated diffusion for hexoses is altered without a decisive impact on the whole system. Moreover, autoradiographic analysis of [14C]galactose-labeled tumor plasma membranes revealed a shift of the incorporation pattern from high- to lower-molecular-weight galactopolypeptides. Our results indicate that specific alterations of the D-galactose metabolism are a characteristic feature of Morris hepatomas.

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane.

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.

Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the rat.

Glycosyltransferase activities of highly purified fractions of Golgi apparatus, plasma membrane and endoplasmic reticulum, all from the same homogenates, were analyzed and compared. Additionally, Golgi apparatus were unstacked and the individual cisternae separated into fractions enriched in cis, median and trans elements using the technique of preparative free-flow electrophoresis. Golgi apparatus from both liver and hepatomas were enriched in all glycosyltransferases compared to endoplasmic reticulum and plasma membranes. However, Golgi apparatus from hepatomas showed both elevated fucosyltransferase and galactosyltransferase activities but reduced sialyltransferase and dipeptidyl peptidase IV (DPP IV) activities compared to liver. Activity of N-acetylglucosaminyltransferase was approximately the same in both liver and hepatoma Golgi apparatus. With normal liver, sialyl- and galactosyltransferase activities and DPP IV showed a marked cis-to-trans gradient of activity. Fucosyltransferase was concentrated in two regions of the electrophoretic separations, one corresponding to cis cisternae and one corresponding to trans cisternae. N-Acetylglucosaminyltransferase activity was more widely distributed but the endogenous acceptor activity was predominantly cis. With hepatoma Golgi apparatus, the pattern for DPP IV was similar to that for liver but those of sialyl- and galactosyltransferases differed markedly from liver. Instead of activity increasing cis to trans, the activities for sialyl- and galactosyltransferases decreased. For fucosyltransferases, activity dependent on exogenous acceptor was medial whereas with endogenous acceptor, two activity peaks, cis and trans, still were observed. For N-acetylglucosaminyltransferase the pattern for hepatoma was similar to that for liver. The results indicate alterations in the distribution of glycosyltransferase activities within the Golgi apparatus in hepatotumorigenesis that may reflect altered cell surface glycosylation patterns.

Uptake, subcellular distribution and metabolism of the phospholipid analogue hexadecylphosphocholine in MDCK cells.

The uptake, subcellular distribution and metabolism of hexadecylphosphocholine was investigated using hexadecylphospho-[methyl-14C]choline as tracer. The phospholipid analogue was taken up in a time-, dose- and temperature-dependent manner and showed a high affinity for the plasma membrane in vivo and in vitro. After incorporation into the plasma membrane, hexadecylphosphocholine appeared in all subcellular membranes investigated but not in the cytosolic fraction. After 24 h, the detectable metabolites of incorporated hexadecylphosphocholine co-chromatographed with the phosphatidylcholine standard (34.3%), the phosphocholine standard (22.1%), the glycerophosphocholine standard (4.3%) and the betaine standard (3.6%).

Effect of chloroquine on the degradation of L-fucose and the polypeptide moiety of plasma membrane glycoproteins.

To evaluate the role of lysosomes in the breakdown of the carbohydrate and the polypeptide moiety of plasma membrane glycoproteins, degradation of the plasma membrane glycoprotein gp120 was studied in the liver of rats treated with the lysosomotropic amine chloroquine. Half-lives of degradation of the terminal sugar L-fucose and of L-methionine of gp120 were measured in isolated plasma membranes after pulse-chase experiments in vivo. Chloroquine extended the plasma membrane half-life of the polypeptide moiety of gp120 from 51 h to 143 h. By contrast, L-fucose of gp120 in the plasma membrane was not affected by chloroquine, but decayed with the same short half-lives of 22 h and 23 h in both controls and chloroquine-treated rats. The data suggest that the protein portion of gp120 is degraded within the lysosomes. Conversely, the terminal sugar L-fucose is removed from the glycoprotein independent from proteolysis before segregation of the glycoprotein into the lysosomal compartment.

Conversion of 14C-galactose into amino acids in tissue culture cells and its inhibition by manganese.

The incorporation of 14C-galactose into primary AGMK-cells was studied in the presence and absence of Mn2+. The transport of galactose into the cells is not influenced by Mn2+. 1 mM MnCl2 inhibits the incorporation of galactose into acid-precipitable material up to 50% after 6 hours incubation. In the absence of Mn2+ a substantial amount of galactose is converted to glucose, which is mainly metabolized into aspartic acid and serine. The conversion of galactose into glucose is inhibited by the addition of Mn2+. However, Mn2+ does not influence the activity of the UDP-galactose-4'-epimerase in vitro. Using the SDS-polyacrylamide electrophoresis the labelling of protein bands is similar with 14C-galactose or a 14C-amino acid mixture, respectively. In the presence of Mn2+ the incorporation of both galactose or amino acids is inhibited: With amino acids the inhibition is observed in all protein bands, whereas with galactose some bands remain unaffected. It is concluded that these are galactoproteins.

Selective isolation of individual cell surface proteins from tissue culture cells by a cleavable biotin label.

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.

Turnover of the protein and carbohydrate moieties of a 105kD glycoprotein of plasma membranes from mouse liver determined by immunoprecipitation.

The turnover of a mouse liver plasma membrane glycoprotein (Mr 105 k) has been studied by means of immunoprecipitation and radioactive labeling. The method of immunoprecipitation proved to be a fast and efficient way of isolating a single protein for measurement of its specific radioactivity. The half-life of the carbohydrate moiety determined using 3H-galactose was 41 h and using 3H-mannose was 23 h, whereas that of the protein moiety determined using 3H-leucine was 63 h. The implications of this differential degradation of the protein and carbohydrate moieties are discussed in terms of the recycling of glycoproteins between the plasma membrane and intracellular membranes.

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes.

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.

Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices.

Protein transport via the endoplasmic reticulum Golgi apparatus-cell surface export route was blocked when slices (6-15 cells thick) of livers of 10-day-old rats were incubated with 1 microM monensin. Production of secretory vesicles by Golgi apparatus was reduced or eliminated and, in their place, swollen cisternae accumulated in the cytoplasm at the trans Golgi apparatus face. The swelling response was restricted to the six external cell layers of the liver slices, and the number of cells showing the response was little increased by either a greater concentration of monensin or by longer times of incubation. When monensin was added post-chase to the slices, flux of radioactive proteins to the cell surface was inhibited by about 80% as determined from standard pulse-chase analyses with isolated cell fractions. Radioactive proteins accumulated in both endoplasmic reticulum and Golgi apparatus and in a fraction that may contain monensin-blocked Golgi apparatus cisternae released from the stack. The latter fraction was characterized by galactosyltransferase/thiamine pyrophosphatase ratios similar to those of Golgi apparatus from control slices. The use of monensin with the tissue slice system may provide an opportunity for the cells to accumulate monensin-blocked Golgi apparatus cisternae in sufficient quantities to permit their isolation and purification by conventional cell fractionation methods.

Vitamin A excess alters membrane flow in rat liver.

Pulse-chase methodology with [35S]methionine as label was employed to determine flow kinetics through the endoplasmic reticulum-Golgi apparatus-(lysosome-) secretory vesicle-plasma membrane export route in livers of animals receiving vitamin A excess by gavage. Overall fraction composition determined by morphometry and by analyses of marker enzymes was unchanged by vitamin administration. The vitamin modified the pattern of flow of proteins through the Golgi apparatus to the cell surface and to lysosomes. Altered flux was evidenced by a markedly reduced rate of labeling of lysosomes and a slightly increased rate of labeling of both total membrane proteins of the plasma membrane and of a specific membrane glycoprotein GP80. Also reduced was overall labeling of the Golgi apparatus. Differences in the rate or routes of trafficking of glycoproteins through the Golgi apparatus together with altered opportunities for processing might account for some of the alterations in glycoconjugate glycosylation associated with excess vitamin A administration.

Phospholipid analogue hexadecylphosphocholine inhibits proliferation and phosphatidylcholine biosynthesis of human epidermal keratinocytes in vitro.

The alkylphospholipid hexadecylphosphocholine (HePC), member of a new class of antineoplastic drugs, has been previously shown to exert cytotoxic effects on neoplastic cell lines in vitro, and a selective antineoplastic activity has been reported after topical application of HePC in vivo, in particular on skin metastases of human mammary carcinomas. Preliminary observations suggest that HePC might also be beneficial in the treatment of non-neoplastic skin diseases characterized by epidermal hyperplasia such as psoriasis. Therefore, we investigated whether HePC might inhibit the proliferation of normal human keratinocytes, and whether its effects might be dependent upon the proliferative status of the treated cells. Moreover, its effects on phosphatidylcholine biosynthesis were studied in keratinocytes. HePC dose-dependently decreased cell numbers, thymidine incorporation, and protein synthesis when applied during the growth phase of keratinocytes grown in serum-free medium, with a minimal inhibitory dose of 10(-7) mol/l for thymidine incorporation, 3 x 10(-7) mol/l for cell numbers, and 10(-6) mol/l for 35S-methionine incorporation. No major differences were observed when keratinocytes were grown under high-Ca++ conditions. In contrast, slowly proliferating confluent keratinocyte cultures showed growth inhibition only after 10(-4) mol/l HePC. Phosphatidylcholine biosynthesis was dose-dependently inhibited by HePC with a half inhibitory concentration of 3 x 10(-6) mol/l, and with translocation of the rate-limiting enzyme. CTP:phosphocholine cytidylyltransferase, to the cytosol, where the enzyme is inactive. These data show a pronounced antiproliferative effect of HePC also on proliferating non-malignant keratinocytes, and are compatible with its possible action on hyperproliferative skin disorders.

Different adhesion inhibiting activities of antisera against plasma membranes of liver and Morris hepatoma 7777.

Cell-substratum adhesion of rat hepatocytes was inhibited by antisera raised against plasma membranes of liver (anti-liver antiserum) and Morris hepatoma 7777 (anti-hepatoma antiserum). Similar concentrations of both antisera inhibited adhesion on collagen. Anti-liver antiserum also inhibited the adhesion of hepatocytes on plastic, whereas anti-hepatoma antiserum was only able to inhibit the adhesion on collagen completely. These results suggest the existence of at least two different adhesion-involved molecules. Cells adhere to plastic by means of both molecules, whereas adhesion on collagen is mediated by only one of them. The results further suggest that hepatoma cells lost the molecule involved in adhesion on plastic.

Identification of a 110-kDa glycoprotein involved in cell-substratum adhesion.

Plasma membrane glycoproteins are involved in cell-matrix interactions. For identification of such glycoproteins a recently developed cell system was used. Two cell populations differing in their adhesion properties were selected from Morris hepatoma 7777. One population was able to grow as a monolayer, while the other proliferated in suspension. From both cell lines spontaneous revertants were selected. By using antibodies raised against plasma membranes of the hepatoma and of rat liver for sequential immunoprecipitation a glycoprotein of Mr 110000 was identified. This glycoprotein was only expressed in adherent hepatoma cells as well as in normal rat liver, but was absent in non-adherent hepatoma cells and in the in vivo growing Morris hepatoma 7777. This suggests that the glycoprotein is involved in cell-substratum adhesion of hepatocytes and adherent hepatoma cells.

Purification of liver and hepatoma membrane proteins by high-performance liquid chromatography.

This paper documents the recovery of selected proteins from hepatic plasma membranes. Initial purification, achieved by a series of stepwise extractions, facilitates the subsequent purification by HPLC. Examples are provided to illustrate the recovery of specific proteins from two Morris hepatoma lines and the liver.

Chemical cross-linking leads to two high molecular mass aggregates of rat alpha 1 beta 1 integrin differing in their conformation but not in their composition.

In order to detect protein interactions of the collagen/laminin receptor alpha 1 beta 1 integrin, covalent chemical cross-linking was performed with the homo-bifunctional, amine reactive reagents DSS (disuccinimidylsuberate) and DSP (dithiobis(succinimidylpropionate)). After cross-linking of the 190 kDa rat alpha 1 integrin subunit, immunoblotting revealed two additional, immunoreactive, high molecular mass complexes (M(r) 240/290 k). Generation of the 240/290 kDa aggregates depended on the presence of the intact tertiary protein structure. As shown with immunoaffinity purified proteins, the 240/290 kDa aggregates consist exclusively of alpha 1 and beta 1 integrin subunits. No other cross-linked proteins associated with the alpha 1 or beta 1 subunit were detected. In contrast to the non-cross-linkable alpha 1 beta 1 integrin, the 240/290 kDa aggregates presumably represent active forms of the adhesion receptor, because both bound in vitro to collagen I and IV. This ability of alpha 1 beta 1 integrin to cross-link and produce two additional high molecular mass forms is shared by rat alpha 9 beta 1 integrin. Thus, the cross-linking approach directly indicates that beta 1 integrins occur in different conformations caused by variations in the folding and/or spatial arrangement of their subunits.