Ingrid Maurer-Fogy

Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.

Cloning and nucleotide sequence of cDNA encoding human erythrocyte band 7 integral membrane protein

cDNA clones encoding the human erythrocyte band 7 membrane protein were isolated by immunoscreening from bone marrow and HeLa cell lambda gt 11 cDNA libraries, and their nucleotide sequences were determined. HeLa- and bone marrow cell-derived sequences were identical, except for one nucleotide; the deduced sequence of 287 amino acids was confirmed by sequence identity with peptides of the erythroid protein. Structure analysis assigned band 7 protein to the type Ib transmembrane proteins.

Vascular anticoagulant beta: a novel human Ca2+/phospholipid binding protein that inhibits coagulation and phospholipase A2 activity. Its molecular cloning, expression and comparison with VAC-alpha.

A cDNA was cloned coding for a new member of the human Ca2+-modulated phospholipid-binding protein family termed annexins. Due to its 56% identity to the human vascular anticoagulant (VAC) the new protein is named VAC-beta, renaming the previous VAC as VAC-alpha. Northern analysis detects one hybridizing mRNA species of 2.2 kb in human placenta. Genomic Southern blot analysis shows a VAC-beta gene of comparable complexity to the VAC-alpha gene. The cDNA was expressed in Escherichia coli and the recombinant protein purified to homogeneity. Antiserum raised against VAC-beta weakly cross-reacts with VAC-alpha. The properties of VAC-beta as an anticoagulant and as an inhibitor of phospholipase A2 activity were analyzed and compared to those of VAC-alpha.

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

Human interferon ω1: isolation of the gene, expression in Chinese hamster ovary cells and characterization of the recombinant protein

A gene encoding human interferon omega-1 (IFN-omega 1) was isolated from a cosmid library, sequenced and expressed in Chinese hamster ovary (CHO) cells under the control of an SV40-derived promoter/enhancer sequence. Culture supernatants of stably transfected cell clones contained biologically active IFN-omega 1 at concentrations up to 10 micrograms/l. Amplification of the expression vector containing a dhfr gene under methotrexate selection pressure resulted in yields up to 200 micrograms/l. Production of IFN-omega 1 was further enhanced 2- to 3-fold by propagation of the cells in the presence of n-butyrate. IFN-omega 1 was purified from culture supernatants by monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase HPLC and size-exclusion HPLC. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed two bands of about the same intensity with apparent molecular masses of 24.5 and 22.5 kDa. Upon treatment with peptide:N-glycosidase F, both bands were shifted to lower molecular masses (20.5 and 18.5 kDa), indicating that CHO cell-derived IFN-omega 1 is glycosylated; Asn-78 was identified as the glycosylation site. Analysis of the carbohydrate moiety using glycosidases and lectins revealed the presence of biantennary complex oligosaccharides containing neuraminic acid. Amino acid sequencing showed that only about 40% of the molecules have the expected N-terminus, whereas the others carry two additional amino acids derived from the signal sequence. C-terminal amino acid sequencing using carboxypeptidase P demonstrated that the smaller form of the protein lacks nine amino acids. Disulfide bridges were shown to connect Cys residues 1 and 99 as well as 29 and 139, respectively, as in IFN-alpha. The specific antiviral activity of recombinant, glycosylated human IFN-omega 1 on human cells was 2.6 x 10(8) IU/mg, not significantly different from that of the authentic, human leukocyte-derived protein.

Natural human tumor necrosis factor beta (lymphotoxin) Variable O‐glycosylation at Thr7, proteolytic processing, and allelic variation

Natural human tumor necrosis factor beta (TNF‐β) purified from supernatants of a human B‐lymphoblastoid cell line was found to be heterogeneous in molecular mass, with seven components resolved by gel electrophoresis. All components are N‐glycosylate at Asn62., N‐glycosylation does not contribute to heterogeneity. In addition, part of the molecules are O‐glycosylated at Thr7; O‐glycosylation is heterogeneous due to variable decoration with neuraminic acid. The four lower molecular mass forms are derived from the full‐length protein by trypsin‐like proteolytic cleavage in the N‐proximal region; these clipped molecules lack O‐linked carbohydrates. Two allelic variants differing in amino acid position 26 (threonine/asparagine) were identified.

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcεRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcεRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(α2–3)Gal(β1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only α-mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.

TRYPSIN SENSITIVITY OF SEVERAL HUMAN RHINOVIRUS SEROTYPES IN THEIR LOW PH-INDUCED CONFORMATION

Five serotypes of human rhinovirus (HRV) were examined for sensitivity to trypsin at physiological pH, HRV1A, HRV2, and HRV14 were found to be resistant whereas in serotypes HRV49 and HRV89 degradation of VP2 was observed. However, exposure to low pH followed by neutralization, a treatment which causes irreversible conformational changes in the capsid, led to rapid cleavage by trypsin of VP1 in HRV1A, HRV2, and HRV49 at defined sites followed by degradation of VP2. In the case of HRV2, the cleavage site in VP1 was determined by direct protein sequencing and was shown to occur between Arg260 and Thr261, close to the C-terminus. HRV49 behaves similarly to HRV2 as expected from extensive sequence similarity in this region, whereas VP1 in HRV1A is most probably cleaved at a site closer to the C-terminus than that in HRV2. Although HRV14 contains the same amino acid pair present in HRV2 and HRV49, it was not cleaved under these conditions. HRV89, which lacks a basic residue at the corresponding position, was also insensitive. Examination of the cleavage site on the three-dimensional structural map of native HRV2 reveals that it is most probably buried inside the capsid and thus not accessible. Structural rearrangements of the viral capsid are thus necessary to account for the cleavage observed after low pH treatment.

Cleavage Site between VP1 and P2A of Human Rhinovirus Is Different in Serotypes 2 and 14

The viral capsid protein VP1 of human rhinovirus serotype 2 (HRV2) was cleaved with cyanogen bromide. The peptides thus obtained were separated on an HPLC butyl reversed phase column. Their positions on VP1 were determined by amino-terminal sequencing using the known nucleotide sequence of the genomic RNA of HRV2. The putative carboxy-terminal peptide was further cleaved with trypsin and the resulting fragments were separated on a C18 reversed phase column. Amino-terminus of sequencing of the C-terminal peptide revealed alanine as being the carboxy terminus of VP1 in HRV2. This indicates that the processing of the polyprotein is different in HRV2 from the processing previously reported for HRV14 and poliovirus.