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Biomedical subjects

H Furthmayr

Publications and source records attributed to H Furthmayr.

At least 91 records · Page 5Linked to original sources
PubMed1981

Mg and Mc: mutations within the amino-terminal region of glycophorin A.

M and N are the two common ("normal") alleles at the MN locus of the MNSs blood group system. The antigens M and N that they determine are located within the amino-terminal region of glycophorin A. In the serologically active and glycosylated (*) fragment of glycophorin AN the sequence is Leu-Ser*-Thr*-Thr*-Glu-, and in that of glycophorin AM it is Ser-Ser*-Thr*-Thr*-Gly-. Mg and Mc are very rare ("variant") alleles of M and N; as to the corresponding antigens, Mg is serologically quite distinct from M and N, while Mc is a compound of both. Erythrocytes of genotypes MgN, MgM, MgMg, and McM, which were the object of the present study, contain normal amounts of glycophorin A in their membrane. In glycophorin AMg the amino-terminal sequence is related to that of glycophorin AN by substitution of asparagine for threonine in position 4, and it is nonglycosylated: Leu-Ser-Thr-Asn-Glu-. The corresponding structure of glycophorin AMc is Ser-Ser*-Thr*-Thr*-Glu-; it is thus closely related to that of glycophorin AN and AM, by substitution of the amino acids in positions 1 or 5, respectively. All of these substitutions can be explained by single base changes. The distinctions in chemical structure not only confirm the location of M and N in this region of glycophorin A, because they are the only differences observed, but also indicate, because they are correlated with the distinctions in antigenic specificity, that M and N are structural genes coding for amino acid sequences. The finding that Mc contains structural features of both M and N suggests that these two forms of glycophorin A have evolved from a common ancestral gene by single base substitutions at sites in the genome coding for amino acids in positions 1 and 5 of the sequence. Carbohydrate structures, however, are also necessary for full expression of antigens M and N. Glycosylation during biosynthesis of residues within the polypeptide appears to depend on a particular protein structure.

Alleles↗
PubMed1981

A network model for the organization of type IV collagen molecules in basement membranes.

Type IV collagen was solubilized from a tumor basement membrane either by acid extraction or by limited digestion with pepsin. The two forms were similar in composition and the size of the constituent chains but differed when examined by electron microscopy and in the fragment pattern produced by bacterial collagenase. The acid-soluble form showed after rotary shadowing strands mainly of a length of 320 nm which terminated in a globule, or two strands connected by a similar globule. The globule was identified as a non-collagenous domain (NC1) which under dissociating conditions could be separated into two peptides showing a monomer-dimer relationship. Higher aggregates of NC1 were visualized under non-dissociating conditions. Some of the acid-extracted molecules have retained the previously 7-S collagen domain. The pepsin-solubilized form lacked domain NC1 and consisted mainly of four triple-helical strands (length 356 nm) joined together at the 7-S domain (length 30 nm). Common to both forms of type IV collagen was a small collagenase-resistant domain NC2 which was composed of collagenous and non-collagenous elements and located between the 7-S domain and the major triple helix. These data indicate that the collagenous matrix of basement membranes consists of a regular network of type IV collagen molecules which is generated by two different interacting sites located at opposite ends of each molecule. The 7-S collagen domain connects four molecules while the NC1 domain connects two molecules. The maximal distance between identical cross-linking sites (7-S or NC1) was estimated to be about 800 nm comprising the length of two molecules.

Animals↗
PubMed1980

Structures of the asparagine-linked sugar chains of glycophorin A.

Glycophorin A isolated from human erythrocytes contains one asparagine-linked sugar chain in one molecule. This sugar chain was successfully released as an oligosaccharide fraction by hydrazinolysis. The oligosaccharide fraction was separated into four components (N, A-1, A-2', and A-2) by paper electrophoresis. Complete structures of the major acidic oligosaccharides (A-1 and A-2) were elucidated by sequential exoglycosidase digestion in combination with methylation study and periodate oxidation as follows: NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(3) [Gal beta 1 leads to 4GlcNAc beta 1 leads to to 2Man alpha 1 leads to 3(6)] (GlcNAc beta 1 leads to 4)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc and NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3) (GlcNAc beta 1 leads to 4)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6) GlcNAc. The abbreviations used are: NeuAc, N-acetylneuraminic acid; GlcNAc, N-acetylglucosamine; Fuc, fucose.

Asparagine↗
PubMed1980

Expression of red cell membrane proteins in erythroid precursor cells.

Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50-60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N-acetyl-phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70-80% of all cells in the spleen of anemic animals, while only 1-2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.

Adult↗
PubMed1980

Collagen polymorphism in the lung. An immunochemical study of pulmonary fibrosis.

The localization of type I, III, IV, and AB2 (V) collagens in normal and fibrotic human lungs was studied by indirect immunofluorescence techniques with affinity purified antibodies specific for these types of collagen. In the normal lung type I collagen appears to be localized to the interstitium of alveolar septa in irregular patterns. Type III appears to have a more prominent, irregular localization in septa and a perivascular localization. Types IV and V codistribute in linear patterns on alveolar and capillary basement membranes. Type V is also present in the interstitium. Dramatic changes in relative amounts and localization were noted in fibrotic lung samples by fluorescence microscopy. In fibrotic lung samples there is a marked increase in type I in thickened septae. Type III is markedly reduced and seen only in perivascular localization. Type V is markedly increased in the interstitium and located in areas of smooth muscle cell proliferation. No apparent change is noted in amount or localization of type IV collagen. These findings correlate well with biochemical data relating to the amounts of various collagens isolated by enzymatic extraction techniques. These results are discussed with respect to staging and evolution of the fibrotic process.

Adult↗
PubMed1980

Codistribution of collagen types IV and AB2 in basement membranes and mesangium of the kidney. an immunoferritin study of ultrathin frozen sections.

Affinity-purified rabbit antibodies specific for collagen types I, III, AB2 and for a partially characterized type IV collagen derived from a murine tumor were used to study the distribution of collagens in the normal mouse kidney. Immunofluorescence staining of conventional frozen sections demonstrated that types I and III were present in bundles around large vessels and in fibers surrounding glomeruli and tubules, whereas types IV and AB2 were distributed in a linear fashion along basement membranes of tubules, glomeruli, and Bowman's capsule and in the mesangial stalk. The distribution of types IV nd AB2 was examined at the ultrastructural level by staining of 600- to 800-A thick frozen sections with a three-stage procedure employing specific collagen antibodies, biotinyl sheep antirabbit IgG, and avidin-ferritin conjugates. Labeling by this procedure demonstrated codistribution of types AB2 and the putative type IV in all three basement membranes. In addition, mesangial matrix was shown to contain both of these collagen types. These results support recent biochemical evidence of collagen heterogeneity in basement membranes, and also support the concept of a structural relationship between mesangial matrix and glomerular basement membranes.

Animals↗
PubMed1980

Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney.

Affinity-purified rabbit antibodies specific for two large noncollagenous gycoproteins--laminin and fibronectin--were used to study the distribution of these proteins in normal murine kidneys. Immunofluorescence staining of conventional frozen sections demonstrates fibronectin within mesangial areas of the glomerulus. Laminin is also found in mesangial areas. However, it also appears to be distributed in typical basement membranelike patterns on glomerular and tubular basement membranes and Bowman's capsule. At the ultrastructural level, by labeling 600-800-A thick frozen sections with a three-stage procedure consisting of specific antibodies, biotinyl sheep anti-rabbit IgG, and avidin-ferritin conjugates, fibronectin is present ony in the mesangial matrix and is specifically localized to areas immediately surrounding mesangial cell processes. Laminin, on the other hand, is found uniformly distributed throughout tubular basement membranes, the mesangial matrix, and Bowman's capsule. In glomerular basement membranes, laminin labeling is restricted to the lamina rara interna and adjacent regions of the lamina densa.

Animals↗
PubMed1980

7-S collagen: characterization of an unusual basement membrane structure.

A new type of collageneous structure, tentatively named 7-S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistance towards further degradation by bacterial collagenase at 20 degrees C. 7-S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl-galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360,000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with tm = 48 degrees C and 70 degrees C. The more stable domain could be isolated as an homogeneous fragment (Mr = 225,000) after a second digestion with collagenase at 37 degrees C. This fragment contained all the disulfide bonds (42 Cys/1,000 residues) of the original molecule. Electron microscopy showed a rod-like structure in agreement with the hydrodynamic properties of 7-S collagen. The dimensions of these peptides were 3 X 95 nm (long form) and 2.4 X 40-50 nm (short form). Complete reduction of 7-S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27,000-153,000 which differ from each other by Mr increments 25,000-27,000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross-linked or represent fragments produced during proteolytic treatments. Complete reduction of 7-S collagen under non-denaturing conditions lowered the thermal transiton of the triple helix to 48 degrees C but did not change its molecular weight except when exposed to dissociating solvents. 7-S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37 degrees C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross-reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7-S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.

Amino Acids↗
PubMed1980

The collagenous components of the subendothelium. Correlation of structure and function.

Endothelial cells provide a continuous nonthrombogenic lining for the vascular tree. Once the endothelium is denuded platelet adhesion and aggregation occur. One postulated mechanism for the nonthrombogenic properties of the endothelial surface is PGI2 production by endothelial cells, which strongly inhibits platelet aggregation. We propose here that the collagen type(s) associated with the endothelial cell surface may also play an important role in these phenomenon. We found that endothelial cells in culture produce types IV and AB2 collagen (both do not aggregate platelets in vitro) and that type AB2 collagen is uniformly distributed on the endothelial cell surface. We have shown this by immunofluorescence microscopy, immunoelectron microscopy, biosynthetic incorporation, and specific immunoprecipitation techniques. Platelets will not aggregate on monolayers of endothelium while they will aggregate on monolayers of other cells cultured from the vascular wall which produce collagen types that aggregate platelets in vitro. Thus, a specific cell surface-associated collagen (type AB2) may be an important determinant in the ability of the endothelial cell to present a nonthrombogenic surface to the blood.

Animals↗
PubMed1979

Induction of erythropoietic colonies in a human chronic myelogenous leukemia cell line.

The ability of cells derived from the K562 cell line to generate erythropoietic colonies was studied. The K562 cell line was derived from a patient with chronic myelogenous leukemia 8 yr ago by Lozzio and Lozzio. Rare benzidine-positive colonies formed when these cells were cloned in plasma clots (3 +/- 1/10(4) cells), and their number was not substantially increased by the addition of erythropoietin (9.5 +/- 1/10(4) cells). Sodium butyrate was capable of markedly enhancing the number of benzidine-positive colonies (19.5 +/- 1/10(4) cells) formed, while the combination of sodium butyrate plus erythropoietin exerted a synergistic effect on erythropoietic colony formation (57 +/- 4/10(4) cells). The K562 cell line is a long-term culture system that contains human erythropoietic stem cells. This cell line should be useful in future studies on the cellular and molecular events associated with human erythroid cell differentiation.

Benzidines↗
PubMed1979

Isolation and tissue localization of type AB2 collagen from normal lung parenchyma.

Type AB2 collagen was isolated from normal lung parenchyma by pepsin extraction followed by differential salt extraction. This collagen comigrates with AB2 collagen isolated from placental membranes when run on 5% polyacrylamide gel electrophoresis; it has an alpha A and alpha B polypeptide chain ratio of 1 : 2 and a cyanogen bromide peptide profile similar to known AB2 collagen on 7.5% polyacrylamide gel electrophoresis. This AB2 collagen isolated from lung tissue specifically inhibits passive hemagglutination of affinity-purified rabbit antibodies to AB2 collagen isolated from amnionic and chorionic membranes. By indirect immunofluorescence microscopy, AB2 collagen was found to be widely distributed throughout the lung and was found preferentially associated with cell surfaces (basement lamina) and basement membranes.

Basement Membrane↗
PubMed1978

Primary structure of human erythrocyte glycophorin A. Isolation and characterization of peptides and complete amino acid sequence.

Peptides of glycophorin AMN were prepared by cyanogen bromide cleavage and by chymotryptic and tryptic digestion. Cyanogen bromide cleavage produces three fragments which account for the entire polypeptide chain. Trypsin and chymotrypsin cleave completely at several sites, but incompletely at sites within the glycosylated segment of the polypeptide chain. Some of the latter sites become accessible to proteolysis after desialation in addition to exposure of new sites for cleavage. The amino acid sequence of glycophorin AMN has been determined by manual Edman degradation, using both the direct Edman and the dansyl-Edman procedures simultaneously for determination of glycosylated amino acid residues. The automated procedure was used for sequence determination of a hydrophobic peptide. Glycophorin A is a polypeptide chain of 131 amino acid residues and contains 16 oligosaccharide units attached to the amino-terminal third of the molecule. Fifteen oligosaccharides are linked O-glycosidically to either threonine or serine residues and one complex oligosaccharide unit is attached N-glycosidically to an asparagine residue. Amino-terminal sequences are different for glycophorin AM and AN, the two forms of the glycophorin A molecule coded for by genes at the MN locus. The differences in sensitivity to proteases of various sites on glycophorin A seem to be due to heterogeneity in the carbohydrate components and not to differences in the primary structure of the polypeptide chains. This work contains a number of revisions and corrections of earlier preliminary reports [Segrest, J.P., Jackson, R. chem. Biophys. Res. Commun, 49, 964-969; Tomita, M., & Marchesi, V.T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964-2968].

Amino Acid Sequence↗
PubMed1978

Structural comparison of glycophorins and immunochemical analysis of genetic variants.

Differences in amino acid sequence of erythrocyte membrane glycophorin A are correlated with M or N blood group activity. A second sialoglycoprotein, glycophorin B, has an amino acid sequence identical to that of glycophorin AN in the first 23 positions and carries N activity only, suggesting that different structural genes code for the glycoproteins carrying these antigens. Certain genetically variant cells lack glycophorin A, as determined by immunochemical methods, and serological MN activity. Other variants lack MN activity, but contain normal amounts of glycophorin A in the membrane.

Amino Acid Sequence↗
PubMed1978

Glycophorins A, B, and C: a family of sialoglycoproteins. Isolation and preliminary characterization of trypsin derived peptides.

Two new sialoglycoproteins, glycophorin B and glycophorin C, were isolated from erythrocyte membranes by extraction with lithium diiodosalicylate, partition in aqueous phenol, gel filtration in detergent, and preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The two proteins were characterized by amino acid and carbohydrate analysis, separation of tryptic peptides, and isolation and purification of the amino terminal glycopeptide from each polypeptide chain. Glycophorin B is found in two forms in electrophoretograms of normal erythrocyte membranes, corresponding to monomer and dimer, as has been similarly described for glycophorin A. By using antibodies to a carboxy terminal determinant of glycophorin A, and direct staining of gels with antibodies and 125I-protein A from Staph. aureus, as well as by two-dimensional immunoelectrophoreis, only the two known forms of glycophorin A are detectable. The data confirm and extend the notion that the sialoglycoproteins in human red cells are dimeric molecules which are either preformed in the membrane or which can readily be generated in vitro. Only glycophorin A and glycophorin C are sensitive to trypsin while in situ in the intact red blood cells.

Amino Acids↗
PubMed1977

31P nuclear magnetic resonance evidence for polyphosphoinositide associated with the hydrophobic segment of glycophorin A.

Glycophorin A, the major human erythrocyte sialoglycoprotein, contains a significant amount of phosphorus when isolated by the lithium diiodosalicylate-phenol procedure. Only a small percentage (approximately 1%) of this phosphorus is phosphoprotein. 31P nuclear magnetic resonance (NMR) analysis of glycophorin A has identified the remaining phosphorus content as phospholipid in origin. From the 31P chemical shifts, the phospholipid has been identified as diphosphoinositide. 31P NMR spectra of the peptides produced by trypsin hydrolysis of glycophorin A reveal that all the diphosphoinositide is closely associated with the hydrophobic region of the protein, suggesting that there is a specific affinity between this phospholipid and the intramembranous portion of glycophorin A.

Binding Sites↗