scholarly journals Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT.

1988 ◽  
Vol 106 (6) ◽  
pp. 1813-1820 ◽  
Author(s):  
J Lipp ◽  
B Dobberstein
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Signal Peptidase ◽  
Membrane Insertion ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Signal Anchor ◽  
Type Ii Membrane Protein

I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal-anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment.

scholarly journals Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein.

1990 ◽  
Vol 111 (1) ◽  
pp. 31-44 ◽  
Author(s):  
M K Spriggs ◽  
P L Collins
Keyword(s):  
Signal Sequence ◽  
Active Form ◽  
Cytoplasmic Domain ◽  
Biologically Active ◽  
Membrane Insertion ◽  
Type I ◽  
Type Ii ◽  
Membrane Anchor ◽  
Amino Terminal ◽  
Signal Anchor

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.

scholarly journals The pulmonary surfactant protein C (SP-C) precursor is a type II transmembrane protein

1991 ◽  
Vol 277 (2) ◽  
pp. 493-499 ◽  
Author(s):  
A Keller ◽  
H R Eistetter ◽  
T Voss ◽  
K P Schäfer
Keyword(s):  
Amino Acid ◽  
Pulmonary Surfactant ◽  
Protein C ◽  
Intracellular Transport ◽  
Transmembrane Protein ◽  
Signal Peptidase ◽  
Type Ii ◽  
Hydrophobic Core ◽  
Amino Acid Residues ◽  
Hydrophobic Domain

Human pulmonary-surfactant-associated protein C (SP-C) is an extremely hydrophobic peptide comprising 34-35 amino acids. It is involved in the reduction of surface tension at the air/liquid in the lung. In order to understand the mechanism by which this molecule is generated from its 197-amino-acid-residues-long precursor and secreted into the alveolar space, we analysed the biosynthesis and processing of this precursor in an ‘in vitro’ system. Our results show that the SP-C precursor is a 21 kDa integral membrane protein. It is anchored in the membrane by a hydrophobic domain that comprises the 20-amino-acid-residues-long hydrophobic core of the mature SP-C peptide. The N-terminus remains in the cytoplasm, which leads to a type II transmembrane orientation of the precursor. Membrane integration occurs in a signal-peptidase-independent manner. The hydrophobic domain acts as both signal sequence and membrane-anchoring domain. We suggest that correct membrane insertion of the SP-C precursor, which is strictly dependent on the hydrophobic-amino-acid sequence represented by the hydrophobic core of the mature SP-C, is itself a prerequisite for further processing and intracellular transport of the mature SP-C.

scholarly journals Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

2015 ◽  
Vol 112 (31) ◽  
pp. 9632-9637 ◽  
Author(s):  
James A. Stapleton ◽  
Timothy A. Whitehead ◽  
Vikas Nanda
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Membrane Proteins ◽  
Cell Membrane ◽  
Membrane Protein ◽  
Computational Design ◽  
Residue Level ◽  
Membrane Insertion ◽  
Amino Acid Residues ◽  
Design Rules

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface ofEscherichia coliOmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus

2006 ◽  
Vol 34 (1) ◽  
pp. 118-121 ◽  
Author(s):  
E.J. Dridge ◽  
D.J. Richardson ◽  
R.J. Lewis ◽  
C.S. Butler
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Archaeoglobus Fulgidus ◽  
Substrate Selectivity ◽  
Type Ii ◽  
Amino Acid Residues ◽  
Sequence Alignments ◽  
X Ray ◽  
Group Type

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.

II. Yeast sequencing reports. The nucleotide sequence ofTTP1, a gene encoding a predicted type II membrane protein

Yeast ◽  
1994 ◽  
Vol 10 (8) ◽  
pp. 1111-1115 ◽  
Author(s):  
Pedro A. Romero ◽  
Ariadni Athanassiadis ◽  
Marc Lussier ◽  
Annette Herscovics
Keyword(s):  
Nucleotide Sequence ◽  
Membrane Protein ◽  
Type Ii ◽  
Gene Encoding ◽  
Type Ii Membrane Protein

scholarly journals Conversion of a class II integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms.

1990 ◽  
Vol 110 (4) ◽  
pp. 999-1011 ◽  
Author(s):  
R G Paterson ◽  
R A Lamb
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Protein ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Protein Fusion ◽  
Native Form ◽  
Hydrophobic Domain ◽  
External Domain ◽  
Signal Anchor

The NH2 terminus of the F1 subunit of the paramyxovirus SV5 fusion protein (fusion related external domain; FRED) is a hydrophobic domain that is implicated as being involved in mediating membrane fusion. We have examined the ability of the FRED to function as a combined signal/anchor domain by substituting it for the natural NH2-terminal signal/anchor domain of a model type II integral membrane protein: the hybrid protein (NAF) was expressed in eukaryotic cells. The FRED was shown to act as a signal sequence, targeting NAF to the lumen of the ER, by the fact that NAF acquired N-linked carbohydrate chains. Alkali fractionation of microsomes indicated that NAF is a soluble protein in the lumen of the ER, and the results of NH2-terminal sequence analysis showed that the FRED is cleaved at a site predicted to be recognized by signal peptidase. NAF was found to be efficiently secreted (t1/2 approximately 90 min) from the cell. By using a combination of sedimentation velocity centrifugation and immunoprecipitation assays using polyclonal and conformation-specific monoclonal antibodies it was found that extracellular NAF consisted of a mixture of monomers, disulfide-linked dimers, and tetramers. The majority of the extracellular NAF molecules were not reactive with the conformation-specific monoclonal antibodies, suggesting they were not folded in a native form and that only the NAF tetramers had matured to a native conformation such that they exhibited NA activity. The available data indicate that NAF is transported intracellularly in multiple oligomeric and conformational forms.

scholarly journals Location of antigenic sites recognized by monoclonal antibodies in the influenza A virus nucleoprotein molecule

2009 ◽  
Vol 90 (7) ◽  
pp. 1730-1733 ◽  
Author(s):  
Natalia L. Varich ◽  
Konstantin S. Kochergin-Nikitsky ◽  
Evgeny V. Usachev ◽  
Olga V. Usacheva ◽  
Alexei G. Prilipov ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Antigenic Variation ◽  
Strain Differences ◽  
Antigenic Structure ◽  
Amino Acid Residues ◽  
Mutant Proteins ◽  
Antigenic Sites

The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.

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