scholarly journals Asparagine-linked oligosaccharides are localized to single extracytosolic segments in multi-span membrane glycoproteins

1994 ◽  
Vol 302 (1) ◽  
pp. 253-260 ◽  
Author(s):  
C Landolt-Marticorena ◽  
R A F Reithmeier
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Na Channel ◽  
Acceptor Site ◽  
Membrane Glycoproteins ◽  
Transmembrane Segments ◽  
Comprehensive Survey ◽  
Single Polypeptide ◽  
Average Size

A comprehensive survey of mammalian multi-span (polytopic) membrane proteins showed that asparagine(N)-linked oligosaccharides are localized to single extracytosolic segments. In most membrane proteins this is because potential consensus sites for N-glycosylation (Asn-Xaa-Ser/Thr, X not equal to Pro) are not found in multiple extracytosolic segments. In functional proteins where consensus N-glycosylation sites are contained within more than one extracytosolic segment, only the first segment contains N-linked carbohydrate. An exception is the alpha-subunit of the Na+ channel, which consists of a duplicated structure containing two glycosylated segments. The average size of established N-glycosylated loops connecting two transmembrane segments is 62 residues, with the smallest glycosylated loop being 33 residues in size. N-glycosylated sites are more highly conserved than non-glycosylated (primarily cytosolic) sites and are more common toward the N-terminus of the membrane domain of multi-span membrane proteins. The optimal conditions for glycosylation of consensus sites within an extracytosolic domain of a multi-span membrane protein are (i) the acceptor site is well-spaced (greater than 10 residues) from the transmembrane domain, (ii) the loop is greater than 30 residues in size and (iii) the segment is the first in the protein to contain a suitable extracytosolic consensus site. The localization of N-linked oligosaccharide chains to a single protein segment suggests either glycosylation of multiple loops may compromise protein folding or function, or only a single polypeptide domain can be optimally glycosylated during biosynthesis in vivo.

scholarly journals Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein

1996 ◽  
Vol 318 (2) ◽  
pp. 645-648 ◽  
Author(s):  
Lisa Y TAM ◽  
Carolina LANDOLT-MARTICORENA ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Band 3 ◽  
Site Directed Mutagenesis ◽  
Acceptor Site ◽  
Oligosaccharyl Transferase ◽  
Transmembrane Segments ◽  
Free Translation ◽  
Reticulocyte Lysates ◽  
Polytopic Membrane Protein

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253–260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.

A new concept in polytopic membrane proteins following from the study of band 3 protein

1998 ◽  
Vol 76 (5) ◽  
pp. 729-733 ◽  
Author(s):  
Naotaka Hamasaki ◽  
Hiroyuki Kuma ◽  
Kazuhisa Ota ◽  
Masao Sakaguchi ◽  
Katsuyoshi Mihara
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Band 3 ◽  
Band 3 Protein ◽  
Transmembrane Peptides ◽  
Lipid Interactions ◽  
Transmembrane Segments ◽  
Novel Concept ◽  
Polytopic Membrane Protein ◽  
Peptide Classification

In the present communication, we introduce a novel concept in multispanning polytopic membrane proteins revealed by the study of the band 3 protein. The transmembrane domain of such proteins can be divided into three categories, that is, hydrophilic loops connecting transmembrane peptides (category 1), portions embedded by peptide-peptide interactions (category 2), and portions embedded by peptide-lipid interactions (category 3). Category 2 peptides of polytopic membrane proteins were found to stably reside in the lipid bilayer without peptide-lipid interactions that had been thought to be essential for transmembrane segments. Category 3 peptides are equivalent to single-spanning segments of bitopic membrane proteins. Three different experiments, namely proteolytic digestion, chemical modification of the band 3 protein, and cell free transcription and translation, were used to categorize the transmembrane peptides.Key words: band 3 protein, transmembrane (TM) peptide, classification of TM, category 2-TM, polytopic membrane protein.

Membrane proteins: from bench to bits

2011 ◽  
Vol 39 (3) ◽  
pp. 747-750 ◽  
Author(s):  
Gunnar von Heijne
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
X Ray ◽  
Protein Topology ◽  
Helix Bundle ◽  
Transmembrane Segments ◽  
Membrane Protein Topology

Membrane proteins currently receive a lot of attention, in large part thanks to a steady stream of high-resolution X-ray structures. Although the first few structures showed proteins composed of tightly packed bundles of very hydrophobic more or less straight transmembrane α-helices, we now know that helix-bundle membrane proteins can be both highly flexible and contain transmembrane segments that are neither very hydrophobic nor necessarily helical throughout their lengths. This raises questions regarding how membrane proteins are inserted into the membrane and fold in vivo, and also complicates life for bioinformaticians trying to predict membrane protein topology and structure.

scholarly journals Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system.

1992 ◽  
Vol 118 (2) ◽  
pp. 245-252 ◽  
Author(s):  
M Yilla ◽  
D Doyle ◽  
J T Sawyer
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Binding Activity ◽  
Translation System ◽  
Folding Intermediates ◽  
Free Translation ◽  
Heterogeneous Interaction ◽  
A Cell ◽  
Reduced State

We previously demonstrated that a heterotypic complex of the two rat asialoglycoprotein receptor subunits was assembled during cell-free translation (Sawyer, J. T., and D. Doyle. 1990. Proc. Natl. Acad. Sci. USA. 87:4854-4858). We have characterized this system further by analyzing polypeptide interactions under both reducing and oxidizing translation conditions. This report shows that the complex represents a heterogeneous interaction between reduced membrane proteins rather than a specific oligomeric structure. In the reduced state membrane proteins interact in this system to form aggregates of diverse size and composition. The aggregated nascent polypeptides interact with the immunoglobulin heavy chain binding protein but this protein is not an integral component of the aggregate. Aggregation occurs via the exoplasmic domain, rather than the transmembrane domain, and the folding of this domain by the formation of intramolecular disulfides, prevents the interaction from occurring. Additionally, the folded molecules containing intramolecular disulfides lack high affinity binding activity and thus appear to resemble the earliest folding intermediates seen in vivo (Olson, J. T., and M. D. Lane. 198. FASEB (Fed. Am. Soc. Exp. Biol.) J. 3:1618-1624). These results lead us to suggest that the formation of intramolecular disulfides during early biogenesis serves to prevent nonspecific associations between nascent polypeptides.

Ethnomedicinal uses, Phytochemistry and Pharmacological Aspects of the Genus Berberis Linn: A Comprehensive Review

2020 ◽  
Vol 23 ◽  
Author(s):  
Roohi Mohi-ud-din ◽  
Reyaz Hassan Mir ◽  
Prince Ahad Mir ◽  
Saeema Farooq ◽  
Syed Naiem Raza ◽  
...  
Keyword(s):  
Chemical Constituents ◽  
Pharmacological Activities ◽  
Traditional Use ◽  
Comprehensive Review ◽  
Wide Range ◽  
Anti Cancer ◽  
Comprehensive Survey ◽  
Ethnomedicinal Uses

Background: Genus Berberis (family Berberidaceae), which contains about 650 species and 17 genera worldwide, has been used in folklore and various traditional medicine systems. Berberis Linn. is the most established group among genera with around 450-500 species across the world. This comprehensive review will not only help researchers for further evaluation but also provide substantial information for future exploitation of species to develop novel herbal formulations. Objective: The present review is focussed to summarize and collect the updated review of information of Genus Berberis species reported to date regarding their ethnomedicinal information, chemical constituents, traditional/folklore use, and reported pharmacological activities on more than 40 species of Berberis. Conclusion: A comprehensive survey of the literature reveals that various species of the genus possess various phytoconstituents mainly alkaloids, flavonoid based compounds isolated from different parts of a plant with a wide range of pharmacological activities. So far, many pharmacological activities like anti-cancer, anti-hyperlipidemic, hepatoprotective, immunomodulatory, anti-inflammatory both in vitro & in vivo and clinical study of different extracts/isolated compounds of different species of Berberis have been reported, proving their importance as a medicinal plant and claiming their traditional use.

scholarly journals Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain

2021 ◽  
Vol 22 (15) ◽  
pp. 7918
Author(s):  
Jisun Hwang ◽  
Bohee Jang ◽  
Ayoung Kim ◽  
Yejin Lee ◽  
Joonha Lee ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Nih3t3 Cells ◽  
C Elegans ◽  
Downstream Signaling ◽  
Downstream Effectors ◽  
Signaling Events

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

scholarly journals NLRP7 deubiquitination by USP10 promotes tumor progression and tumor-associated macrophage polarization in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Bing Li ◽  
Zhi-Peng Qi ◽  
Dong-Li He ◽  
Zhang-Han Chen ◽  
Jing-Yi Liu ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Acceptor Site ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Chemokine Ligand ◽  
Specific Protease ◽  
Ubiquitin Specific Protease ◽  
Chemokine Ligand 2

Abstract Background NOD-like receptors affect multiple stages of cancer progression in many malignancies. NACHT, LRR, and PYD domain-containing protein 7 (NLRP7) is a member of the NOD-like receptor family, although its role in tumorigenesis remains unclear. By analyzing clinical samples, we found that NLRP7 protein levels were upregulated in colorectal cancer (CRC). We proposed the hypothesis that a high level of NLRP7 in CRC may promote tumor progression. Here, we further investigated the role of NLRP7 in CRC and the underlying mechanism. Methods NLRP7 expression in human CRC and adjacent non-tumorous tissues was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effect of NLRP7 in CRC progression was investigated in vitro and in vivo. Proteins interacting with NLRP7 were identified by immunoprecipitation and mass spectrometry analysis while immunofluorescence staining revealed the cellular location of the proteins. Cellular ubiquitination and protein stability assays were applied to demonstrate the ubiquitination effect on NLRP7. Cloning and mutagenesis were used to identify a lysine acceptor site that mediates NLRP7 ubiquitination. Cytokines/chemokines affected by NLRP7 were identified by RNA sequencing, qRT-PCR, and enzyme-linked immunosorbent assay. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and immunohistochemistry. Results NLRP7 protein levels, but not mRNA levels, were upregulated in CRC, and increased NLRP7 protein expression was associated with poor survival. NLRP7 promoted tumor cell proliferation and metastasis in vivo and in vitro and interacted with ubiquitin-specific protease 10, which catalyzed its deubiquitination in CRC cells. NLRP7 stability and protein levels in CRC cells were modulated by ubiquitination and deubiquitination, and NLRP7 was involved in the ubiquitin-specific protease 10 promotion of tumor progression and metastasis in CRC. K379 was an important lysine acceptor site that mediates NLRP7 ubiquitination in CRC cells. In CRC, NLRP7 promoted the polarization of pro-tumor M2-like macrophages by inducing the secretion of C-C motif chemokine ligand 2. Furthermore, NLRP7 promoted NF-κB nuclear translocation and activation of C-C motif chemokine ligand 2 transcription. Conclusions We showed that NLRP7 promotes CRC progression and revealed an as-yet-unidentified mechanism by which NLRP7 induces the polarization of pro-tumor M2-like macrophages. These results suggest that NLRP7 could serve as a biomarker and novel therapeutic target for the treatment of CRC.

scholarly journals A Novel Golgi Membrane Protein Is a Partner of the ARF Exchange Factors Gea1p and Gea2p

2003 ◽  
Vol 14 (6) ◽  
pp. 2357-2371 ◽  
Author(s):  
Sophie Chantalat ◽  
Rëgis Courbeyrette ◽  
Francesca Senic-Matuglia ◽  
Catherine L. Jackson ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Trafficking ◽  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Membrane Dynamics ◽  
General Interest ◽  
Single Mutation ◽  
Golgi Membrane ◽  
Nucleotide Exchange

The Sec7 domain guanine nucleotide exchange factors (GEFs) for the GTPase ARF are highly conserved regulators of membrane dynamics and protein trafficking. The interactions of large ARF GEFs with cellular membranes for localization and/or activation are likely to participate in regulated recruitment of ARF and effectors. However, these interactions remain largely unknown. Here we characterize Gmh1p, the first Golgi transmembrane-domain partner of any of the high-molecular-weight ARF-GEFs. Gmh1p is an evolutionarily conserved protein. We demonstrate molecular interaction between the yeast Gmh1p and the large ARF-GEFs Gea1p and Gea2p. This interaction involves a domain of Gea1p and Gea2p that is conserved in the eukaryotic orthologues of the Gea proteins. A single mutation in a conserved amino acid residue of this domain is sufficient to abrogate the interaction, whereas the overexpression of Gmh1p can compensate in vivo defects caused by mutations in this domain. We show that Gmh1p is an integral membrane protein that localizes to the early Golgi in yeast and in human HeLa cells and cycles through the ER. Hence, we propose that Gmh1p acts as a positive Golgi-membrane partner for Gea function. These results are of general interest given the evolutionary conservation of both ARF-GEFs and the Gmh proteins.

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