Membrane binding mechanism of an RNA virus-capping enzyme.

The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation. NMR structure shows that the alpha-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.

Ultrasensitive time-resolved immunofluorometric assay of glutathione transferase alpha in serum.

Glutathione transferase Alpha (GSTA) is a very sensitive marker of acute centrilobular liver damage. We purified glutathione transferases from human liver and separated the isoenzymes. Polyclonal rabbit antibodies specific for Alpha-class isoenzymes were produced and labeled with Eu(3+)-chelate. We set up a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) to measure GSTA in serum. The detection limit was 0.03 microgram/L, and measuring range was from 0.03 to 100 micrograms/L. The reference range for serum GSTA in this assay was 0.7-6.0 micrograms/L for women and 0.7-14 micrograms/L for men. This TR-IFMA is practical, and the modified rapid assay can be completed in 3 h. Therefore it is applicable for diagnostic purposes in acute liver damage, e.g., associated with drug toxicity or hypoperfusion of the liver.

Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34.

Monoclonal antibodies of the CD34 class all recognize a monomeric cell surface antigen of approximately Mr 110,000 which is selectively expressed on human hemopoietic progenitor cells. This structure can be readily surface-labeled with [125I]actoperoxidase and by periodate-[3H]borohydride, but it labels only weakly with [35S]methionine, [35Sl]cysteine, 3H-amino acids, or 3H-mannose, even after prolonged labeling periods. However, the antigen is more efficiently labeled by [3H]glucosamine. Lectin binding studies, sensitivity to certain glycosidases, and gel filtration analysis of glycans released by alkaline hydrolysis indicate that this glycoprotein contains several complex-type N-linked glycans as well as several highly sialylated O-linked glycans. Western blotting experiments show that various CD34 antibodies fail to efficiently detect desialylated and/or de-N-glycosylated forms of the antigen. Experiments involving the use of tunicamycin, together with metabolic labeling studies, strongly suggest that this structure "turns over" very slowly in vivo. The CD34 antigen is not detectably labeled by 32P-phosphate in vivo, nor are immune complexes containing it associated with phosphokinase activity in vitro. Sequential immunoprecipitation and Western blotting studies indicate that this antigen is not a member of the leukosialin/sialophorin family despite the fact that these molecules share several structural similarities. Partial amino acid analysis of highly purified CD34 antigen revealed no significant sequence similarity with any previously described structures.

The correlation of breakpoint cluster region rearrangement and p210 phl/abl expression with morphological analysis of Ph-negative chronic myeloid leukemia and other myeloproliferative diseases.

The chromosome 22 derivative, the Philadelphia (Ph) chromosome, results from a reciprocal translocation t(9;22) (q34;q11) and is associated with chronic myeloid leukemia (CML). The translocation can be identified at the DNA level in Ph-positive CML by using a probe to the breakpoint cluster region (bcr). In addition, as a result of this translocation an abl-related 210-kd protein with protein tyrosine kinase (PTK) activity is produced. We analyzed 28 cases of Ph-negative CML for rearrangement of the chromosome 22 sequences and found that eight of the 28 show rearrangement of the bcr. When 12 of the Ph-negative cases were independently reviewed, five were indistinguishable from Ph-positive CML on the basis of morphology, peripheral blood film and clinical details. These five also showed bcr rearrangement. The other seven were reclassified as six atypical CML (aCML) and one chronic myelomonocytic leukemia (CMML). None of these seven showed bcr rearrangement. In addition 11 cases of bcr- CML were assayed for abl-related PTK, and no detectable activity was present, whereas p210 phl/abl PTK was observed both in Ph-positive (three cases examined) and Ph-negative, bcr + (four cases examined) CML. Therefore, bcr + CML, whether or not the Ph chromosome is cytogenetically apparent, involves a similar molecular alteration and produces the 210-kd protein with enhanced PTK activity. Furthermore, these cases can be distinguished from Ph-negative bcr- CML by careful evaluation of clinical and hematologic data.

Distribution and epitope analysis of the cell membrane glycoprotein (HPCA-1) associated with human hemopoietic progenitor cells.

Monoclonal antibodies My10, BI.3C5, 12.8, and ICH3 identify a monomeric cell surface glycoprotein (HPCA-1) of 100-120 kD, which is selectively expressed on human hemopoietic progenitor cells. Other tissues are nonreactive with the exception of capillary endothelia and basement membrane in some sites. In addition, the antigen can be detected on cell lines that exhibit characteristics associated with early T cell precursors. HPCA-1 is therefore associated with myeloid, B, and T lineage precursors. Sequential immunoprecipitation and Western blotting studies demonstrate that BI.3C5, ICH3, My10, and an antibody directed against endothelial cells, 188.27, all react with the same glycoprotein species, although the epitopes involved may be distinct. The epitope recognized by BI.3C5 is sialic acid dependent, whereas that recognized by ICH3 is not. The My10 epitope has partial sensitivity to neuraminidase. Competitive/additive binding experiments suggest that these epitopes, although probably distinct, may be closely associated.

A novel abl protein expressed in Philadelphia chromosome positive acute lymphoblastic leukaemia.

The Philadelphia (Ph) chromosome breakpoints in chronic myelocytic leukaemia are clustered on chromosome 22 band q11 in a 5.8-kilobase (kb) region designated bcr. The c-abl protooncogene is translocated from chromosome 9 band q34 into bcr and the biochemical consequence of this molecular rearrangement is the production of an abnormal fusion protein bcr-abl p210 with enhanced protein-tyrosine kinase activity compared to the normal p145 c-abl protein. The Ph chromosome translocation is also seen in some acute lymphoblastic leukaemias with B-cell precursor phenotypes some of which have bcr rearrangement (bcr+) and some do not (bcr-). We present evidence that the Ph+, bcr- leukaemias are associated with a novel p190 abl kinase. We propose that acute lymphoblastic leukaemias that are bcr+, p210+ are probably lymphoid blast crises following a clinically silent chronic phase of chronic myelocytic leukaemia arising in multipotential stem cells whereas bcr-, p190+ cases are de novo acute lymphoblastic leukaemias arising in more restricted precursors.

Molecular biology of Philadelphia chromosome in chronic granulocytic leukaemia and acute lymphoblastic leukaemia.

In classical t(9;22) translocation, as observed in chronic granulocytic leukemia (CGL), a hybrid DNA unit is produced, including a rearranged PHL gene, previously known as bcr (breakpoint cluster region) plus the translocated c-abl gene from chromosome 9: a hybrid bcr-abl protein, p210 is formed, with increased tyrosine kinase activity. Such DNA rearrangement, with a p210 protein synthesis, is also found in cases of Philadelphia-positive acute lymphoblastic leukemia (ALL), but in apparently similar cases the bcr gene is not rearranged, and a novel p190 abl-related protein can be found; c-abl rearrangement has also been observed.It is thus established that correlations between cytogenetic and molecular events can be found in CGL and ALL, as in other haemopoietic malignancies: translocation and possible rearrangement of the c-abl oncogene seem of particular importance in this case.

Lectin-binding sites in normal, hyperplastic, adenomatous and carcinomatous human colorectal mucosa.

The carbohydrate structures of cellular glycoconjugates in normal, hyperplastic, adenomatous and carcinomatous human colorectal mucosa were analysed with six fluorescein isothiocyanate-conjugated lectins. In normal, hyperplastic and adenomatous colorectal mucosa showing mild or moderate dysplasia Concanavalin A (Con A), Lens culinaris (LCA), and wheat germ (WGA) agglutinins stained goblet cell glycoconjugates (actual mucin goblet itself) while peanut (PNA), Vicia villosa (VVA), and Griffonia simplicifolia-II (GSA-II) agglutinins showed a supranuclear staining of goblet cell glycoconjugates. After neuraminidase treatment of tissue sections PNA and VVA stained mucin goblets of mature cells in normal mucosa, while less differentiated cells in the lower crypt displayed a supranuclear staining with VVA. The mucin goblets in adenomatous mucosa with mild or moderate dysplasia did not stain with PNA and VVA, neither before nor after neuraminidase treatment. Areas of in situ cancer in adenomas and carcinomas displayed a strong and direct binding of Con A, LCA, WGA and PNA in an apical linear distribution, while the binding of VVA and GSA-II was heterogeneous. We conclude that there are alterations in the carbohydrate structures of cellular glycoconjugates, which can be related to goblet cell differentiation in normal colorectal mucosa and to the degree of dysplasia in adenomas. Heterogeneous and incompletely glycosylated glycoconjugates appear to be synthesized by the majority of colorectal carcinomas.

Antiserum against formalin-fixed human milk fat globule glycoprotein for immunohistochemistry of normal and malignant apocrine epithelium.

A rabbit antiserum was raised against formalin-fixed glycoprotein isolated by lectin affinity chromatography from human milk fat globules. After adsorption with erythrocytes and normal blood leukocytes the antiserum detected in immunoblotting of fresh mammary carcinoma tissue a major component of 75 000 dalton apparent molecular weight. The antiserum specifically decorated normal and malignant apocrine epithelium in sections of formalin-fixed tissues. The usefulness of such antisera for routine immunohistochemical diagnosis of metastatic carcinomas is demonstrated.

Association of Rho(D) polypeptides with the membrane skeleton in Rho(D)-positive human red cells.

The Rho(D) antigen was recently identified as a 28,000 to 33,000 m.w. polypeptide expressed on the surface of human Rho(D)+ cells. We now show that 70 to 80% of the Rho(D) polypeptides remain firmly associated with the membrane skeleton (detergent-insoluble matrix) obtained after treatment of isolated membranes with Triton X-100. The same treatment solubilized most of the major sialoglycoprotein, glycophorin A. The membrane skeleton-bound Rho(D) polypeptides were not solubilized by procedures that dissociated spectrin, actin, and glyceraldehyde-3-phosphate dehydrogenase from the membrane. Affinity-purified 125I-labeled anti-Rho(D) antibodies bound to intact Rho(D)+ cells, Rho(D)+ membranes, and isolated membrane skeletons from Rho(D)+ cells, but not to Rho(D)- cells. The binding to Rho(D)+ cells was competitively inhibited efficiently by Rho(D)+ membranes and weakly by Rho(D)- membranes. When isolated unsealed Rho(D)+ and Rho(D)- membranes were labeled by lactoperoxidase-catalyzed iodination and solubilized in Triton X-100, Rho(D) polypeptides were immune precipitated only from Rho(D)+ membranes.

Biosynthesis of the major human red cell sialoglycoprotein, glycophorin A. A review.

The human leukemia cell line K562 is erythroid and expresses the major red cell sialoglycoprotein, glycophorin A. With this cell line we have studied the biosynthesis of glycophorin A after pulse-chase labeling with [35S] methionine. Using lectin-Sepharose affinity chromatography and immune precipitation with specific anti-glycophorin A antiserum followed by polyacrylamide slab gel electrophoresis a precursor of glycophorin A was visualized. This had an apparent molecular weight of 37000 and contained an incompleted N-glycosidic oligosaccharide and unfinished O-glycosidic oligosaccharides. After chase for 10 min, the completed glycophorin A with an apparent molecular weight of 39000 was seen and it appeared at the cell surface in about 30 min. Using tunicamycin N-glycosylation was inhibited but not O-glycosylation. The absence of the N-glycosidic oligosaccharide did not affect the migration of the protein to the cell surface but the yield of glycophorin A was diminished. Translation of glycophorin A messenger-RNA was achieved in a rabbit reticulocyte cell-free system. This yielded a non-glycosylated protein with an apparent molecular weight of 19500, which exceeded that of the glycophorin A apoprotein with about 5000. This indicates the presence of a "signal sequence" in the preprotein. When the translation was performed in the presence of microsomal membranes from dog pancreas the glycophorin A apoprotein aws both N-and O-glycosylated and the apparent molecular weight (37000) of the synthesized protein was identical to that of the precursor obtained from cells.

Expression of blood group A antigens in human bone marrow cells.

We have studied the appearance of blood group A-activity during hematopoiesis in human bone marrow cells by the use of the blood group A-specific lectin from Vicia cracca. Cells that bound the lectin were identified using antiserum against the lectin followed by rosetting with protein A-containing Staphylococcus aureus cells. Only cells of the erythroid lineage from blood group A individuals formed staphylococcal rosettes. A-activity occurred in basophilic normoblasts and later stages of erythropoiesis, whereas pronormoblasts were negative. The appearance of blood group A-activity coincided roughly with the onset of hemoglobin synthesis and slightly later than the expression of the major sialoglycoprotein of erythrocytes, glycophorin A. Glycophorin A did not, however, contain blood group A-activity when analyzed by immunoprecipitation and gel electrophoresis.

Blood-group A and B determinants are located in different polyglycosyl peptides isolated from human erythrocytes of blood-group AB.

The distribution of blood-group A and B determinants was studied by isolating blood-group ABH-active polyglycosyl peptides from delipidated human blood-group AB erythrocyte membranes after extensive digestion with pronase followed by chromatography on Bandeiraea simplicifolia I (BsI) lectin coupled to Sepharose. 20% of the polyglycosyl peptides were bound to BsI lectin. The glycopeptides bound were further fractionated using the blood-group-A-specific lectin from Vicia cracca (Vc). Approximately half of these were bound to the Vc lectin. The glycopeptides, which were bound to the Vc column, were not bound to the blood-group-B-specific isolectin from B. simplicifolia (BsIB4) whereas the Vc-unbound glycopeptides readily bound. The results indicate that in the polyglycosyl peptides isolated from AB erythrocytes A and B determinants are located in different carbohydrate chains. The polyglycosyl peptides, which did not bind to BsI lectin, were composed on the average of 30 monosaccharide units and those that bound contained on the average 55 monosaccharide units. The sugar composition was similar in both fractions except that N-acetylgalactosamine was found only in the BsI-bound glycopeptides. The substitution patterns of the monosaccharides were quite similar in both fractions except 2,3-O-linked galactose, which was enriched 7.5-fold in the BsI-bound glycopeptides and 3,6-O-linked galactose, which also enriched in the BsI-bound glycopeptides suggesting that these have a more branched structure than the BsI-unbound glycopeptides. Glycopeptides derived from bands 3 and 4.5 were prepared from A1B-blood-group erythrocyte membranes and fractionated as above. 25% of the glycopeptides were bound to BsI-lectin from both samples. 70% of the BsI-bound material from band 3 was bound to Vc lectin and 60% from band 4.5. The results indicate heterogeneity in the glycosylation of these bands.