scholarly journals Secretion of a type II integral membrane protein induced by mutation of the transmembrane segment

1997 ◽  
Vol 322 (1) ◽  
pp. 335-342 ◽  
Author(s):  
Isabelle LEMIRE ◽  
Claude LAZURE ◽  
Philippe CRINE ◽  
Guy BOILEAU
Keyword(s):  
Helical Structure ◽  
Structural Data ◽  
Neutral Endopeptidase ◽  
Integral Membrane Protein ◽  
Full Length ◽  
Signal Peptidase ◽  
Soluble Form ◽  
Type Ii ◽  
Α Helix ◽  
Flanking Regions

Signal peptide/membrane anchor (SA) domains of type II membrane proteins initiate the translocation of downstream polypeptides across the endoplasmic reticulum (ER) membrane. In contrast with signal peptides, however, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncated (without cytoplasmic domain) protein were constructed by substitution of the sequences SQNS, QQTT or YPGY for VTMI starting at position 15 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of these sequences was decided from our previous observation that substitution in the NEP SA domain of the sequence SQNS, which is polar and has α-helix-breaking potential, could promote SA domain processing under certain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268, 2699Ő2704; Yang, Chatellard, Lazure, Crine and Boileau (1994) Arch. Biochem. Biophys. 315, 382Ő386). The QQTT sequence is polar but, according to secondary structure predictions, is compatible with the α-helix structure of the NEP SA domain. The YPGY sequence and single Pro residue are less polar and have α-helix-breaking potential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptides encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-1 cells and their fate (membrane-bound or secreted) was determined by immunoblotting and by endoglycosidase digestions. Our biochemical and structural data indicate that: (1) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) α-helix-breaking residues are not a prerequisite for cleavage; (3) the presence, in close proximity to a putative signal peptidase cleavage site, of a polar sequence that maintains the α-helical structure of the SA domain is sufficient to promote cleavage. Furthermore pulseŐchase studies suggest that cleavage is performed in the ER by signal peptidase and indicate that cleavage is not a limiting step in the biosynthesis of the soluble form of the protein.

scholarly journals Borrelia burgdorferi BBA74, a Periplasmic Protein Associated with the Outer Membrane, Lacks Porin-Like Properties

2006 ◽  
Vol 189 (5) ◽  
pp. 2063-2068 ◽  
Author(s):  
Vishwaroop Mulay ◽  
Melissa J. Caimano ◽  
Dionysios Liveris ◽  
Daniel C. Desrosiers ◽  
Justin D. Radolf ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Borrelia Burgdorferi ◽  
Outer Membrane ◽  
Membrane Vesicle ◽  
Integral Membrane Protein ◽  
Proteinase K ◽  
Signal Peptidase ◽  
Signal Peptidase I ◽  
Membrane Spanning ◽  
Α Helix

ABSTRACT The outer membrane of Borrelia burgdorferi, the causative agent of Lyme disease, contains very few integral membrane proteins, in contrast to other gram-negative bacteria. BBA74, a Borrelia burgdorferi plasmid-encoded protein, was proposed to be an integral outer membrane protein with putative porin function and designated as a 28-kDa outer membrane-spanning porin (Oms28). In this study, the biophysical properties of BBA74 and its subcellular localization were investigated. BBA74 is posttranslationally modified by signal peptidase I cleavage to a mature 25-kDa protein. The secondary structure of BBA74 as determined by circular dichroism spectroscopy consists of at least 78% α-helix with little β-sheet structure. BBA74 in intact B. burgdorferi cells was insensitive to proteinase K digestion, and indirect immunofluorescence microscopy showed that BBA74 was not exposed on the cell surface. Triton X-114 extraction of outer membrane vesicle preparations indicated that BBA74 is not an integral membrane protein. Taken together, the data indicate that BBA74 is a periplasmic, outer membrane-associated protein that lacks properties typically associated with porins.

scholarly journals Expression of an enzymically active glycosylphosphatidylinositol-anchored form of neutral endopeptidase (EC 3.4.24.11) in Cos-1 cells

1994 ◽  
Vol 299 (1) ◽  
pp. 171-176 ◽  
Author(s):  
S Howell ◽  
C Lanctôt ◽  
G Boileau ◽  
P Crine
Keyword(s):  
Cell Surface ◽  
Cell Types ◽  
Neutral Endopeptidase ◽  
Integral Membrane Protein ◽  
Cellular Transport ◽  
Type Ii ◽  
Wild Type ◽  
Form Expression ◽  
Km Value ◽  
Frame Fusion

Neutral endopeptidase (EC 3.4.24.11, NEP) is a type-II integral membrane protein found in a wide variety of cell types. We previously produced a secreted form of the enzyme by deletion of the cytoplasmic and transmembrane domains and in-frame fusion of the cleavable signal peptide of pro-opiomelanocortin [Lemay, Waksman, Roques, Crine and Boileau (1989) J. Biol. Chem. 264, 15620-15623]. Here we have used this secreted form of NEP and fused to it the glycosylphosphatidylinositol (GPI)-anchor attachment signal of decay-accelerating factor to produce a GPI-anchored form. Expression of this chimeric form in Cos-1 cells resulted in cell-surface activity. This activity could be released from the cell surface by phosphatidylinositol-specific phospholipase C and radiolabelling studies showed that the protein could incorporate [3H]ethanolamine, indicating that the enzyme was GPI-anchored. The Km value, using [D-Ala2,Leu5]enkephalin as substrate, of GPI-anchored NEP (62 +/- 5 microM) was comparable with that of wild-type NEP (70 +/- 4 microM), as were the sensitivities to the inhibitors phosphoramidon and thiorphan. However, pulse-chase studies showed that the biosynthesis and cell-surface delivery of GPI-anchored NEP was delayed compared with that of the wild-type transmembrane form of NEP. These results suggest a lower rate of biosynthesis and/or cellular transport for GPI-anchored NEP compared with its transmembrane counterpart.

scholarly journals The nature of topogenic sequences determines the transport competence of topological mutants of neutral endopeptidase-24.11

1995 ◽  
Vol 312 (1) ◽  
pp. 99-105 ◽  
Author(s):  
X F Yang ◽  
P Crine ◽  
G Boileau
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Neutral Endopeptidase ◽  
Soluble Form ◽  
Type I ◽  
Type Ii ◽  
Mutant Proteins ◽  
Endopeptidase 24.11 ◽  
C Terminus ◽  
Cytosolic Domains

Type II integral membrane proteins are anchored by a signal-peptide/membrane-anchor domain (SA domain) located near their N-terminus, whereas type I membrane proteins are anchored by stop-transfer sequences usually located near the C-terminus. In this study we have attempted to transform neutral endopeptidase-24.11 (EC 3.4.24.11; NEP), a type II membrane protein, into a type I membrane protein. Three type I mutant proteins were constructed by fusion of topogenic sequences to the C-terminus of SecNEP, a soluble form of NEP. The first two type I mutants, SecNEP-TMC and SecNEP-TMIC, were constructed by fusing in frame the cytosolic and SA domains of NEP to the C-terminus of SecNEP. These two fusion proteins differ only in the orientation of the cytosolic tail. The third type I mutant, SecNEP-ACE, was constructed by fusing in frame the stop-transfer and cytosolic domains of angiotensin I-converting enzyme (EC 3.4.15.1; ACE) to the C-terminus of SecNEP. Our results suggest that: (1) the NEP ectodomain can be anchored with a type I topology in the endoplasmic reticulum (ER) membrane by both NEP and ACE topogenic sequences; (2) SecNEP-TMC and SecNEP-TMIC were transport-incompetent and needed proteolytic cleavage in the C-terminal region to leave the ER, whereas SecNEP-ACE was transported out of the ER as a type I membrane protein. Therefore we concluded that the nature of topogenic sequences determines the transport-competence of topological mutants of neutral endopeptidase-24.11.

scholarly journals Maturation ofStreptococcus pneumoniaelipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence

2008 ◽  
Vol 67 (3) ◽  
pp. 541-557 ◽  
Author(s):  
Suneeta Khandavilli ◽  
Karen A. Homer ◽  
Jose Yuste ◽  
Shilpa Basavanna ◽  
Timothy Mitchell ◽  
...  
Keyword(s):  
Abc Transporter ◽  
Signal Peptidase ◽  
Type Ii

scholarly journals Structural and functional analysis of the Na+/H+ exchanger

2007 ◽  
Vol 401 (3) ◽  
pp. 623-633 ◽  
Author(s):  
Emily R. Slepkov ◽  
Jan K. Rainey ◽  
Brian D. Sykes ◽  
Larry Fliegel
Keyword(s):  
Intracellular Ph ◽  
Sequence Similarity ◽  
Structural Data ◽  
Structural Similarity ◽  
Integral Membrane Protein ◽  
Volume Control ◽  
Amino Acid Residues ◽  
Physiological Processes ◽  
High Resolution Structure ◽  
Extracellular Sodium

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.

scholarly journals Cysteine Mutagenesis Reveals Novel Structure–Function Features within the Predicted Third Extracellular Loop of the Type Iia Na+/Pi Cotransporter

2001 ◽  
Vol 117 (6) ◽  
pp. 533-546 ◽  
Author(s):  
Georg Lambert ◽  
Ian C. Forster ◽  
Gerti Stange ◽  
Katja Köhler ◽  
Jürg Biber ◽  
...  
Keyword(s):  
Structure Function ◽  
Helical Structure ◽  
Cysteine Residue ◽  
Transport Function ◽  
Extracellular Loop ◽  
Substituted Cysteine Accessibility ◽  
Novel Structure ◽  
Important Region ◽  
Α Helix ◽  
Low Sensitivity

The transport function of the rat type IIa Na+/Pi cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a “structure–function” profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an α-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated α-helical structure.

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 Tubular Assembly Formation Induced by Leucine Alignment along the Hydrophobic Helix of Amphiphilic Polypeptides

2021 ◽  
Vol 22 (21) ◽  
pp. 12075
Author(s):  
Mohammed A. Abosheasha ◽  
Toru Itagaki ◽  
Yoshihiro Ito ◽  
Motoki Ueda
Keyword(s):  
Double Layer ◽  
Self Assembly ◽  
Helical Structure ◽  
Tube Formation ◽  
Rolled Sheet ◽  
Α Helix ◽  
Necessary And Sufficient ◽  
Hydrophobic Helix ◽  
Nanotube Dispersion ◽  
Spherical Vesicles

The introduction of α-helical structure with a specific helix–helix interaction into an amphipathic molecule enables the determination of the molecular packing in the assembly and the morphological control of peptide assemblies. We previously reported that the amphiphilic polypeptide SL12 with a polysarcosine (PSar) hydrophilic chain and hydrophobic α-helix (l-Leu-Aib)6 involving the LxxxLxxxL sequence, which induces homo-dimerization due to the concave–convex interaction, formed a nanotube with a uniform 80 nm diameter. In this study, we investigated the importance of the LxxxLxxxL sequence for tube formation by comparing amphiphilic polypeptide SL4A4L4 with hydrophobic α-helix (l-Leu-Aib)2-(l-Ala-Aib)2-(l-Leu-Aib)2 and SL12. SL4A4L4 formed spherical vesicles and micelles. The effect of the LxxxLxxxL sequence elongation on tube formation was demonstrated by studying assemblies of PSar-b-(l-Ala-Aib)-(l-Leu-Aib)6-(l-Ala-Aib) (SA2L12A2) and PSar-b-(l-Leu-Aib)8 (SL16). SA2L12A2 formed nanotubes with a uniform 123 nm diameter, while SL16 assembled into vesicles. These results showed that LxxxLxxxL is a necessary and sufficient sequence for the self-assembly of nanotubes. Furthermore, we fabricated a double-layer nanotube by combining two kinds of nanotubes with 80 and 120 nm diameters—SL12 and SA2L12A2. When SA2L12A2 self-assembled in SL12 nanotube dispersion, SA2L12A2 initially formed a rolled sheet, the sheet then wrapped the SL12 nanotube, and a double-layer nanotube was obtained.

scholarly journals Biophysical characterization of the SARS-CoV-2 E protein

2021 ◽  
Author(s):  
Aujan Mehregan ◽  
Sergio Perez-Conesa ◽  
Yuxuan Zhuang ◽  
Ahmad Elbahnsi ◽  
Diletta Pasini ◽  
...  
Keyword(s):  
Recombinant Expression ◽  
Structural Data ◽  
Md Simulations ◽  
Full Length ◽  
Coarse Grained ◽  
Biophysical Characterization ◽  
Viral Budding ◽  
Nmr Structures ◽  
E Protein

SARS-CoV-2 is the virus responsible for the COVID-19 pandemic which continues to wreak havoc across the world, over a year and a half after its effects were first reported in the general media. Current fundamental research efforts largely focus on the SARS-CoV-2 Spike protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length E protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

scholarly journals Expression of Functional CD32 Molecules on Human NK Cells Is Determined by an Allelic Polymorphism of the FcγRIIC Gene

Blood ◽  
1998 ◽  
Vol 91 (7) ◽  
pp. 2369-2380 ◽  
Author(s):  
Diana Metes ◽  
Linda K. Ernst ◽  
William H. Chambers ◽  
Andrei Sulica ◽  
Ronald B. Herberman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Null Allele ◽  
Nk Cell ◽  
Full Length ◽  
Null Alleles ◽  
Soluble Form ◽  
Allelic Polymorphism ◽  
Reading Frame ◽  
Normal Individuals ◽  
Alternatively Spliced

Human natural killer (NK) cells were thought to express only FcγRIIIA (CD16), but recent reports have indicated that NK cells also express a second type of FcγR, ie, FcγRII (CD32). We have isolated, cloned, and sequenced full-length cDNAs of FcγRII from NK cells derived from several normal individuals that may represent four different products of the FcγRIIC gene. One transcript (IIc1) is identical with the already described FcγRIIc form. The other three (IIc2-IIc4) appear to represent unique, alternatively spliced products of the same gene, and include a possible soluble form. Analyses of the full-length clones have revealed an allelic polymorphism in the first extracellular exon, resulting in either a functional open reading frame isoform or a null allele. Stable transfection experiments enabled us to determine a unique binding pattern of anti-CD32 monoclonal antibodies to FcγRIIc. Further analyses of NK-cell preparations revealed heterogeneity in CD32 expression, ranging from donors lacking CD32 expression to donors expressing high levels of CD32 that were capable of triggering cytotoxicity. Differences in expression were correlated with the presence or absence of null alleles. These data show that certain individuals express high levels of functional FcγRIIc isoforms on their NK cells.

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