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K Drickamer

Publications and source records attributed to K Drickamer.

At least 91 records · Page 5Linked to original sources
PubMed1987

Major and minor forms of the rat liver asialoglycoprotein receptor are independent galactose-binding proteins. Primary structure and glycosylation heterogeneity of minor receptor forms.

Preparations of the rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL), which is responsible for the selective uptake of partially deglycosylated serum glycoproteins, have been found to contain multiple polypeptide species. A method has been developed for separating the predominant species (RHL-1) from the minor species (RHL-2/3) using conditions in which RHL-1 retains its galactose-binding activity. Endoglycosidase digestion and lectin blotting have been utilized to demonstrate that RHL-2 and RHL-3 differ by the presence of different carbohydrate structures attached to a common peptide backbone. Antisera specific for RHL-1 and RHL-2/3 have been prepared and utilized to analyze the results of cross-linking experiments performed on purified receptor and hepatocyte membranes. The results show that RHL-1 and RHL-2/3 polypeptides are each associated into homooligomers but are physically unlinked to each other. The structure of the RHL-2/3 polypeptide has been established by protein and cDNA sequence analysis, which reveals that this protein is homologous to RHL-1 throughout its length but contains one major insertion of 18 amino acids near its NH2 terminus. The COOH-terminal portion of the RHL-2/3 polypeptide has been demonstrated to contain a galactose-recognition domain by expression in an in vitro transcription/translation system. The results of these experiments indicate that RHL-1 and RHL-2/3 polypeptides are self-associated into two distinct molecules, each of which has galactose-binding activity.

Amino Acid Sequence↗
PubMed1987

The chicken receptor for endocytosis of glycoproteins contains a cluster of N-acetylglucosamine-binding sites.

The oligomeric state of the chicken hepatic receptor for N-acetylglucosamine-terminated glycoproteins (the chicken hepatic lectin) has been examined in detergent solution, in various membrane preparations, and in hepatocytes. In detergent solution, the cross-linking reagent, 1,5-difluoro-2,4-dinitrobenzene produces covalent complexes containing up to six receptor polypeptides. This result, along with hydrodynamic studies of the receptor-detergent complex, indicates that the purified receptor is a hexamer. Analysis of large proteolytic fragments of the receptor reveals that portions of the receptor polypeptide near the membrane anchor are essential for hexamer stability. This analysis also demonstrates that each receptor polypeptide has an N-acetylglucosamine-binding site, indicating that the native hexameric receptor contains a cluster of six such sites. Immunoblot analysis of membrane fractions and cells cross-linked with 1,5-difluoro-2,4-dinitrobenzene or dimethyl adipimidate reveals that the receptor is also oligomeric in intact cells and in subcellular fractions representing cell surface and internalized receptor. Although the pattern of cross-linking observed in membranes differs from that observed with purified receptor, experiments indicate that the differences may be explained by the presence of membrane components which compete with receptor for reaction with cross-linking reagent. The presence of a cluster of carbohydrate-binding sites in the hepatocyte membrane can account for the preferential endocytosis of multivalent glycoprotein ligands by hepatocytes.

Acetylglucosamine↗
PubMed1987

Exon structure of a mannose-binding protein gene reflects its evolutionary relationship to the asialoglycoprotein receptor and nonfibrillar collagens.

Cloned cDNAs encoding mannose-binding proteins isolated from rat liver have been used to isolate one of the genes encoding this group of proteins. This gene, which encodes the minor form of binding protein (designated MBP-A), has been characterized by sequence analysis. The protein-coding portion of the mRNA for the MBP-A is encoded by four exons separated by three introns. The NH2-terminal, collagen-like portion of the protein is encoded by the first two exons. These exons resemble the exons found in the genes for nonfibrillar collagens in that the intron which divides them is inserted between the first two bases of a glycine codon and the exons do not have the 54- or 108-base pair lengths characteristic of fibrillar collagen genes. The carbohydrate-binding portion of MBP-A is encoded by the remaining two exons. This portion of the protein is homologous to the carbohydrate-recognition domain of the hepatic asialoglycoprotein receptor, which is encoded by four exons. It appears that the three COOH-terminal exons of the asialoglycoprotein receptor gene have been fused into a single exon in the MBP-A gene. The organization of the MBP-A gene is very similar to the arrangement of the gene encoding the highly homologous pulmonary surfactant apoprotein, although one of the intron positions is shifted by a single amino acid. The 3' end of a mannose-binding protein pseudogene has also been characterized.

Amino Acid Sequence↗
PubMed1987

The stereospecificity of protein kinases.

To test whether cellular protein kinases exist that phosphorylate D-amino acid residues, a method was developed for separating O-phospho-D-serine from O-phospho-L-serine and O-phospho-L-tyrosine from O-phospho-D-tyrosine. This was accomplished by converting these amino acids to the L-leucyl dipeptide derivatives followed by separation of the diastereomers by anion-exchange high-performance liquid chromatography. The enantiomeric content of these D- and L-residues were measured in hydrolysates of 32P-labeled proteins produced by the protein kinases of human erythrocytes and the tyrosyl protein kinase of the Abelson leukemia virus. We found no measurable D-phosphoserine in erythrocyte membrane proteins under conditions where a 1% content of this residue relative to L-phosphoserine would have been detected. These values can be used to place an upper hypothetical limit on the fraction of erythrocyte protein kinase activity that is specific for serine residues in the D-configuration. In separate experiments, we examined the specificity of the tyrosyl protein kinases. We found that all of the phosphotyrosine that we isolated from the erythrocyte band 3 NH2-terminal fragment and from the autophosphorylation of the Abelson virus tyrosyl kinase was in the L-configuration.

Blood Proteins↗
PubMed1986

Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein.

Preparations of mannose-binding protein isolated from rat liver contain two distinct but homologous polypeptides. The complete primary structures of both of these polypeptides have been determined by sequencing of peptides derived from the proteins, isolation and sequencing of cDNAs for both proteins, and partial characterization of the gene for one of the proteins. Each polypeptide consists of three regions: (a) an NH2-terminal segment of 18-19 amino acids which is rich in cysteine and appears to be involved in the formation of interchain disulfide bonds which stabilize dimeric and trimeric forms of the protein, (b) a collagen-like domain consisting of 18-20 repeats of the sequence Gly-X-Y and containing 4-hydroxyproline residues in several of the Y positions, and (c) a COOH-terminal carbohydrate-binding domain of 148-150 amino acids. The sequences of the COOH-terminal domains are highly homologous to the sequence of the COOH-terminal carbohydrate-recognition portion of the chicken liver receptor for N-acetylglucosamine-terminated glycoproteins and the rat liver asialoglycoprotein receptor. Each protein is preceded by a cleaved, NH2-terminal signal sequence, consistent with the finding that this protein is found in serum as well as in the liver. The entire structure of the mannose-binding proteins is homologous to dog pulmonary surfactant apoprotein.

Amino Acid Sequence↗
PubMed1986

The rat liver asialoglycoprotein receptor polypeptide must be inserted into a microsome to achieve its active conformation.

Affinity chromatography on galactose-Sepharose has been utilized to demonstrate that rat liver asialoglycoprotein receptor synthesized in vitro in a reticulocyte lysate system is capable of binding carbohydrate ligand only when dog pancreas microsomes are present during translation. Analysis of receptor isolated from tunicamycin-treated rat hepatocytes indicates that glycosylation is not necessary for receptor activity. Genetically engineered receptor derivatives in which the natural membrane anchor is either deleted entirely or replaced with a cleavable signal sequence derived from dog preproinsulin have been used to demonstrate that: (a) inactive receptor made in the absence of membranes does not result from incorrect nucleation of folding around the hydrophobic portion of the polypeptide which is normally buried in the membrane and (b) the carbohydrate-binding domain of the receptor does not need to be tethered to the luminal side of the membrane to fold correctly. These results suggest that factors within the lumen of the microsomes are essential to establish the native conformation of the binding domain.

Acetylglucosaminidase↗
PubMed1986

Signal recognition particle mediates the insertion of a transmembrane protein which has a cytoplasmic NH2 terminus.

The mechanism by which rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL) is inserted into membranes has been investigated. RHL is a prototype for transmembrane proteins which are oriented with their NH2 termini in the cytoplasm and their COOH termini outside the cell. Such transmembrane proteins are synthesized without cleavable NH2-terminal signal sequences. An in vitro translation system has been developed in which RHL is translated from RNA produced in a bacteriophage SP6 in vitro transcription system. RHL produced in this way can be inserted cotranslationally in the correct orientation into dog pancreas microsomes. This insertion process has been shown to be dependent on the signal recognition particle and its receptor (the docking protein) in the microsomal membranes. A detailed mechanism for the insertion of this type of transmembrane protein into the lipid bilayer is proposed.

Amino Acid Sequence↗
PubMed1985

Characterization of the gene encoding the major rat liver asialoglycoprotein receptor.

A cloned cDNA encoding the major rat liver asialoglycoprotein receptor has been used to analyze the gene for this protein. Genomic Southern blot analysis reveals that the gene is contained on a single EcoRI restriction fragment and is unique. A clone containing the gene (isolated from a rat liver genomic library) has been characterized by sequence analysis. The mRNA for the receptor is encoded by nine exons separated by eight introns. The first exon is confined to the 5'-untranslated region of the mRNA, the second exon encodes most of the cytoplasmic NH2-terminal domain of the receptor polypeptide, the third exon corresponds to the hydrophobic transmembrane portion of the polypeptide, and the remaining exons encode the extracellular parts of the receptor. Some, but not all, of the divisions between exons correspond to boundaries between functional domains of the polypeptide.

Animals↗
PubMed1984

Direct evidence for the transmembrane orientation of the hepatic glycoprotein receptors.

The technique of vectorial labeling has been used to study the orientation of the rat and chicken receptors for asialo- and agalactoglycoproteins in hepatocyte membranes. The membrane-impermeant enzyme lactoperoxidase was used to radioiodinate the outer surfaces of intact cells and endocytic vesicles, as well as both sides of total microsomal membranes. Proteolytically and chemically produced fragments of the receptor polypeptides were analyzed to identify the tyrosine residues modified in each case. The results reveal that each of these receptors is a transmembrane glycoprotein arranged with its NH2 terminus facing the cytoplasm and its COOH terminus, containing the carbohydrate-binding site, exposed at the cell surface. While the primary structures of the chicken and rat receptors are highly homologous in the extracellular portions of the proteins, the cytoplasmic domains show no sequence similarity.

Animals↗
PubMed1984

Primary structure of the rat liver asialoglycoprotein receptor. Structural evidence for multiple polypeptide species.

When preparations of rat liver receptor for asialoglycoproteins (rat hepatic lectin, (RHL] are examined by dodecyl sulfate-polyacrylamide gel electrophoresis, multiple polypeptide species are found to be present. The predominant polypeptide has an apparent molecular weight of 41,500 (RHL-1), while two less abundant species appear to be of higher molecular weight (49,000 (RHL-2) and 54,000 (RHL-3]. When the several polypeptides are separated and treated with BrCN, the two minor species are found to share at least one large fragment, while the RHL-1 species gives rise to a completely different set of BrCN peptides. All of the BrCN fragments of the major species and the large common fragment from RHL-2 and RHL-3 have been isolated. These fragments serve as the basis for the complete sequence determination of RHL-1. The complete sequence is 283 residues long, although 20% of the protein as isolated is missing the first 2 residues at the NH2 terminus. The overall arrangement of the polypeptide is similar to the chicken receptor for asialoagalactoglycoproteins; it consists of an NH2-terminal stretch of hydrophilic amino acids, a segment of about 30 uncharged residues, and a COOH-terminal portion which contains three oligosaccharide attachment sites. When the COOH terminus of the rat liver receptor is aligned with the corresponding portions of the chicken liver receptor, the two proteins show 28% identity. Little identity is seen near the NH2 terminus. Sequence homology between residues 50-79 and residues 121-150 of the rat receptor suggests that the additional length of this protein compared with the chicken protein may be due to the presence of a duplicated segment within the rat receptor. The complete sequence of the BrCN fragment common to the two minor species has also been determined; this 101-residue sequence is 53% identical with the COOH-terminal sequence of RHL-1. Since these minor species have a primary structure distinct from RHL-1, there must be at least two genes coding for receptor polypeptides. RHL-2 and RHL-3 may differ in their extent of posttranslational modification.

Amino Acid Sequence↗
PubMed1984

Rat liver asialoglycoprotein receptor lacks a cleavable NH2-terminal signal sequence.

Two cDNA clones encoding the predominant form of the asialoglycoprotein receptor from rat liver (the major rat hepatic lectin; RHL-1) were identified by screening a rat liver cDNA library with a mixed oligonucleotide probe 35 nucleotides long. One clone was a nearly full-length copy of the mRNA for RHL-1, while the other was shortened at both ends. The sequences of these clones demonstrate that this transmembrane receptor is not synthesized with an NH2-terminal signal sequence. The only proteolytic processing occurring in the biosynthesis of RHL-1 is the removal of the NH2-terminal initiator methionine residue. Insertion of RHL-1 into the membrane is postulated to occur by the recognition of the internal transmembrane region as a signal sequence.

Amino Acid Sequence↗
PubMed1983

The primary structure of the cytotoxin alpha-sarcin.

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.

Amino Acid Sequence↗
PubMed1983

A tyrosine kinase associated with the red cell membrane phosphorylates band 3.

Phosphorylation of Band 3, the anion transport protein of human erythrocyte membranes, has been studied by incubating isolated ghosts with [gamma-32P]ATP. One of the phosphate-acceptor sites is tyrosine 8 in the NH2-terminal cytoplasmic domain of the Band 3 protein. Seven out of 11 residues in the sequence surrounding the phosphorylated tyrosine are Asp or Glu. It is concluded that the erythrocyte, like other cells, contains a membrane-associated tyrosine kinase which phosphorylates highly anionic peptide acceptor sites.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1982

Phosphorylation of a membrane receptor for glycoproteins. Possible transmembrane orientation of the chicken hepatic lectin.

The chicken hepatic lectin, a receptor for partially deglycosylated serum glycoproteins, has been identified as a phosphoprotein. Phosphorylation was detected by incorporation of 32P into the protein in cultured hepatocytes and by two-dimensional gel analysis of protein purified from liver tissue. In addition, forms of the receptor containing one, two, and three sialic acid residues have been detected, with the disialylated form predominating. The site of phosphorylation has been identified as Ser7 in the complete amino acid sequence of the receptor (Drickamer, K. (1981) J. Biol. Chem. 256, 5827-5839). The presence of a protein kinase target site near the NH2-terminal of the receptor, a stretch of 25 uncharged, hydrophobic residues in positions 24 through 48, and a site of glycosylation at position 67 suggests that the chicken hepatic lectin is probably a transmembrane protein, oriented with COOH-terminal outside the cell and NH2-terminal in the cytoplasm.

Amino Acid Sequence↗
PubMed1981

Complete amino acid sequence of a membrane receptor for glycoproteins. Sequence of the chicken hepatic lectin.

The chicken hepatic lectin is involved in the clearance of glycoproteins from circulation (Kawasaki, T., and Ashwell, G. (1977) J. Biol. Chem. 252, 6536-6543). The complete amino acid sequence of chicken hepatic lectin has been established by analysis of peptides generated by chemical cleavage at methionine or tryptophan residues. Larger BrCN fragments were further digested with trypsin, chymotrypsin, and clostripain. All sequences were determined by automated sequential Edman degradation. Extensive use was made of high performance liquid chromatography in the purification of peptides and identification of phenylthiohydantoin derivatives of amino acids. The complete sequence is: (formula: see text). The stretch of uncharged amino acids from residue 25 to 48 is a possible membrane-interaction region. Carbohydrate is attached to residue 67.

Amino Acid Sequence↗
PubMed1981

Amino acid sequence of the precursor of rat liver alpha 2 micro-globulin.

The amino acid sequence of the signal peptide of the rat liver protein alpha 2 micro-globulin has been determined using a combination of nucleic acid and protein sequencing techniques. The NH2-terminal portion of pre-alpha 2 micro-globulin is: (formula see text) As is the case with most secreted proteins, the majority (13 of 19) of the amino acid residues in this signal are hydrophobic. However, there are no obvious similarities between this signal sequence and that determined for preproalbumin, a protein presumably synthesized and secreted by the same cells in the liver.

Alpha-Globulins↗