Consensus N-linked Glycosylation Sites Research Articles

Primary structure of the mouse [formula omitted] receptor gene

The mouse β 1- adrenergic receptor was isolated from a genomic library and cloned into pBluescript SK −. Characterization of the clone revealed an open reading frame which encodes a predicted protein of 466 amino acids. The mouse β 1 receptor is 92.7% identical to the human sequence, 98.5% identical to the rat sequence, and contains a consensus site for N-linked glycosylation at Asn-15 and a cAMP-dependent protein kinase phosphorylation site at Ser-301.

Brugia pahangi and Brugia malayi: A Surface-Associated Glycoprotein (gp15/400) Is Composed of Multiple Tandemly Repeated Units and Processed from a 400-kDa Precursor

The cuticle of filarial nematode parasites contains distinct and separable sets of soluble and structural proteins. Surface-labeling techniques have previously identified a soluble protein complex in adult stage Brugia which ranges in molecular weight from 15 to 200 kDa. Using an antiserum directed to the 15-kDa basal subunit of this complex, we show here that this complex is synthesized and processed from a single, very large precursor protein with a molecular weight of approximately 400 kDa. Molecular cloning, sequencing, and Southern analysis indicates that the protein is encoded by a single gene composed predominantly of approximately 20 tandemly repeated segments of 396 bp. The two complete copies of these repeated segments in our cDNA sequence are identical. Each subunit of 132 amino acids bears a consensus site for N-linked glycosylation, and glycosidase treatment indicates that this corresponds to an oligosaccharide side chain of 2 kDa. The protein displays no significant homology to sequences lodged in databases corresponding to molecules of known function. Nevertheless, a significant similarity (19/41 residues) is observed with the N-terminal sequence of a protein termed ABA-1, an allergen from Ascaris.

Molecular cloning and sequence of a novel ommochrome-binding protein cDNA from an insect, Manduca sexta.

An ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta has recently been purified and characterized. A cDNA clone was isolated from a fifth instar larval cDNA expression library utilizing antiserum against OBP. Northern blot analysis of total fat body RNA detected a transcript of approximately 1.2 kilobases in fifth instar wandering larvae RNA. The complete nucleotide sequence of the 905-base pair cDNA insert was determined by the dideoxy chain termination method. The OBP cDNA encodes a polypeptide of 274 residues with a predicted molecular weight of 30,580 and with one consensus N-linked glycosylation site. Comparison of the NH2-terminal sequence of the mature protein and the cDNA sequence revealed a typical signal peptide of 18 amino acids. In wandering stage larvae, the OBP transcript appeared to be at least 250-fold less abundant than ribosomal RNA.

Human Skin Mast Cell Carboxypeptidase: Functional Characterization, cDNA Cloning, and Genealogy

We functionally characterized human skin mast cell carboxypeptidase A (MC-CPA), and explored its evolutionary relationship to other carboxypeptidases to understand further the structural basis for the substrate preferences of this enzyme. Purified human skin MC-CPA displayed more activity than did bovine pancreatic carboxypeptidase A (CPA) against carboxyl-terminal leucine residues, about equal activity with phenylalanine and tyrosine residues, and no activity with tryptophan or alanine. To correlate kinetic data with structure, we isolated and sequenced a cDNA encoding MC-CPA from human skin, and directly sequenced 30% of the purified protein. These sequences agreed with that of human lung MC-CPA, and further support the evidence for a single MC-CPA gene in humans. Four amino acid replacements, resulting in a net positive change in non-hydrogen atoms in the S1' subsite of MC-CPA, were associated with less alteration in substrate specificity, relative to bovine CPA, than might be expected from studies using rat CPA1 and CPA2. We noted two consensus N-linked glycosylation sites in human MC-CPA that are not found in rat and mouse MC-CPA, or in bovine CPA; that at least one of these sites is glycosylated in vivo was verified by N-glycosidase F treatment, lentil lectin binding, and Concanavalin A-Sepharose chromatography. Evolutionary trees constructed from the known carboxypeptidase sequences suggested that MC-CPA most likely evolved from a carboxypeptidase B-like enzyme, independent of the pancreatic CPA. Thus, in the carboxypeptidase gene family, MC-CPA displays a unique genealogy and several amino acid replacements in its S1' binding pocket that result in substrate specificity quite similar to bovine CPA.

Nonrandom distribution of gp120 N-linked glycosylation sites important for infectivity of human immunodeficiency virus type 1.

More than 20 consensus N-linked glycosylation sites occur in the gp120 coding sequence of most isolates of human immunodeficiency virus type 1. Based on the N-linked glycosylation pattern of a well-characterized recombinant gp120, it is likely that N-linked sugars are present at most, if not all, of the consensus glycosylation sites of the heavily glycosylated gp120. In this study, we evaluated the relative importance of each of the 24 N-linked glycosylation sites of gp120 in the molecular clone HXB2 to viral infectivity. The ability of HXB2-derived mutants, each having 1 of the 24 N-linked glycosylation sites mutated by site-directed mutagenesis, to infect CD4-positive SupT1 cells was compared with that of the wild-type virus. We found that most of the individual consensus N-linked glycosylation sites are dispensable for viral infectivity. The five consensus N-linked glycosylation sites that are likely to have important roles in infectivity are all located in the amino-terminal half of gp120, indicating that the N-linked glycosylation sites that are important for infectivity of human immunodeficiency virus type 1 are not randomly distributed in gp120. We predict that a partially glycosylated gp120 with most of the dispensable N-linked glycosylation sites removed may be a better vaccine candidate than the fully glycosylated gp120.

Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.

Using the sequence homology approach for cloning related genes within the G-protein-coupled receptor gene family, we have cloned the gene for the rat beta 1-adrenergic receptor (beta 1-AR). The rat beta 1-adrenergic receptor gene was isolated from a lambda EMBL3 rat genomic DNA library using the hamster beta 2-adrenergic receptor (beta 2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat beta 1-AR gene encodes a protein of 466 amino acids that contains one consensus site for N-linked glycosylation (Asn-15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (Ser-296, Ser-301, and Ser-401). The encoded rat beta 1-AR is 98 and 91% similar at the amino acid level with the human beta 1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat beta 1-AR gene is a single copy gene. The tissue distribution of the rat beta 1-AR mRNA was highest in the pineal gland with other brain regions and peripheral tissues, including the heart, expressing the mRNA at moderate levels. The bacteriophage clone containing the rat beta 1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into beta 1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the beta 1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the beta 1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki approximately 140 times lower than that for the beta 2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by beta-adrenergic receptor agonists with the rank order of potency of isoproterenol greater than norepinephrine = epinephrine, consistent with properties expected of the beta 1-AR subtype.

Molecular characterization of a rat alpha 2B-adrenergic receptor.

Alpha 2-adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. We have isolated a cDNA clone (pRNG alpha 2) encoding a rat alpha 2-adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNG alpha 2 DNA display high affinity and saturable binding to [3H]rauwolscine (Kd = 2 nM). Competition curve data analysis shows that RNG alpha 2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine greater than or equal to chlorpromazine greater than or equal to prazosin greater than or equal to clonidine greater than norepinephrine greater than or equal to oxymetazoline. RNG alpha 2 RNA accumulates in both rat kidney and neonatal rat lung (predominant species is 4000 nucleotides). When a cysteine residue (Cys-169) that is conserved among all members of the seven-transmembrane-region superfamily is changed to phenylalanine, the RNG alpha 2 protein fails to bind [3H]rauwolscine after expression in COS cells. We conclude that pRNG alpha 2 likely represents a cDNA for a rat alpha 2B-adrenergic receptor.

Neutralizing epitopes of lymphocytic choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression

Lymphocytic choriomeningitis virus (Armstrong strain) bears two overlapping epitopes, GP-1A (A) and GP-1D (D), recognized by neutralizing antibodies on the major surface glycoprotein GP-1. Both are discontinuous conformational epitopes that require prior formation of disulfide bridges and addition of N-linked oligosaccharides. Using monoclonal antibodies specific for each of these epitopes, as well as for conformation-independent epitopes, we have investigated the requirements for biosynthesis and folding of the epitopes. The carbohydrate residues themselves do not appear to comprise critical informational components of these epitopes, but are required for proper folding of the nascent glycopeptide chain within the rough endoplasmic reticulum. These epitopes differ in their resistance to denaturation; epitope D is retained when denatured with SDS under nonreducing conditions, whereas epitope A is lost. Monoclonal antibodies to epitope A cross-react with several strains of LCMV. However, epitope D is detected in only a subset of isolates derived from the Armstrong strain of LCMV. By RNA sequence analysis, we have mapped a single amino acid change distinguishing those virions containing epitope D. Acquisition of binding activity of the epitope D-specific monoclonal correlates with a Thr→Ala or Thr→Lys mutation at amino acid 173 of the GP-1 molecule and concomitant disruption of a consensus N-linked glycosylation site.