scholarly journals tANCHOR: a novel mammalian cell surface peptide display system

BioTechniques ◽  
2020 ◽  
Author(s):  
Daniel Ivanusic ◽  
Kazimierz Madela ◽  
Heidi Burghard ◽  
Gudrun Holland ◽  
Michael Laue ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Amino Acid Sequences ◽  
Display System ◽  
Extracellular Loop ◽  
Binding Studies ◽  
Small Proteins ◽  
Potential Applications ◽  
Large Extracellular Loop ◽  
Peptide Display

A novel tool for the presentation of peptides and small proteins on the surface of human cells has been developed. Our tANCHOR system utilizes tetraspanin anchors containing heterologous amino acid sequences inserted instead of the large extracellular loop. This technology allows a highly effective extracellular display of epitopes for antibody binding studies and many other potential applications.

Proteins on the surface of the malaria parasite and cell invasion

Parasitology ◽  
1994 ◽  
Vol 108 (S1) ◽  
pp. S5-S18 ◽  
Author(s):  
A. A. Holder
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Malaria Parasite ◽  
Surface Protein ◽  
Cell Types ◽  
Merozoite Surface Protein ◽  
Amino Acid Sequences ◽  
Specific Cell ◽  
Binding Studies ◽  
Erythrocyte Invasion

SUMMARYThe malaria parasite exists in an extracellular form at several stages in its life cycle. Within the vertebrate host, sporozoites and merozoites have to invade specific cell types. Proteins on the surface of the parasite or externalized from specialized organelles have been implicated as ligands for receptors on the host cell surface. Direct binding studies have identified parasite proteins that interact with the target cell surface. Examination of the deduced amino acid sequences has allowed the identification of primary structural motifs which may have roles in this process. On the sporozoite, the circum-sporozoite protein and sporozoite surface protein-2, a protein initially located within micronemes, have been found to contain an amino acid sequence thought to be involved in mediating recognition of sulphated polysaccharides on the surface of a liver cell. On the merozoite, merozoite surface protein-1 may be involved in the initial recognition of red blood cells; this protein undergoes a complex series of modifications in the time between its synthesis as a precursor molecule and successful erythrocyte invasion. Other merozoite proteins located at the apical end of the parasite have been identified as erythrocyte or reticulocyte binding proteins.

scholarly journals The amino acid sequences of the carboxyl termini of human and mouse hepatic lipase influence cell surface association

2003 ◽  
Vol 44 (7) ◽  
pp. 1306-1314 ◽  
Author(s):  
Robert J. Brown ◽  
Joshua R. Schultz ◽  
Kerry W. S. Ko ◽  
John S. Hill ◽  
Tanya A. Ramsamy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Hepatic Lipase ◽  
Amino Acid Sequences ◽  
Human And Mouse

scholarly journals Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail.

1990 ◽  
Vol 111 (3) ◽  
pp. 955-966 ◽  
Author(s):  
M A Williams ◽  
M Fukuda
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Chimeric Protein ◽  
Cytoplasmic Tail ◽  
Amino Acid Sequences ◽  
Lysosomal Membrane ◽  
Cdna Clones ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Membrane Glycoproteins

Human lysosome membrane glycoprotein h-lamp-1 is a highly N-glycosylated protein found predominantly in lysosomes, with low levels present at the cell surface. The signal required for delivery of h-lamp-1 to lysosomes was investigated by analyzing the intracellular distribution of h-lamp-1 with altered amino acid sequences expressed from mutated cDNA clones. A cytoplasmic tail tyrosine residue found conserved in chicken, rodent, and human deduced amino acid sequences was discovered to be necessary for efficient lysosomal transport of h-lamp-1 in COS-1 cells. In addition, the position of the tyrosine residue relative to the membrane and carboxyl terminus also determined lysosomal expression. Supplanting the wild-type h-lamp-1 cytoplasmic tail onto a cell surface reporter glycoprotein was sufficient to cause redistribution of the chimera to lysosomes. A similar chimeric protein replacing the cytoplasmic tyrosine residue with an alanine was not expressed in lysosomes. Altered proteins that were not transported to lysosomes were found to accumulate at the cell surface, and unlike wild-type lysosomal membrane glycoproteins, were unable to undergo endocytosis. These data indicate that lysosomal membrane glycoproteins are sorted to lysosomes by a cytoplasmic signal containing tyrosine in a specific position, and the sorting signal may be recognized both in the trans-Golgi network and at the cell surface.

scholarly journals Mapping of the CXCR4 Binding Site within Variable Region 3 of the Feline Immunodeficiency Virus Surface Glycoprotein

2008 ◽  
Vol 82 (18) ◽  
pp. 9134-9142 ◽  
Author(s):  
Magnus Sundstrom ◽  
Rebecca L. White ◽  
Aymeric de Parseval ◽  
K. Jagannadha Sastry ◽  
Garrett Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Feline Immunodeficiency Virus ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Surface Glycoprotein ◽  
Binding Studies ◽  
Virus Surface ◽  
Immunodeficiency Virus ◽  
Feline Model ◽  
V3 Region

ABSTRACT Feline immunodeficiency virus (FIV) shares with T-cell tropic strains of human immunodeficiency virus type 1 (HIV-1) the use of the chemokine receptor CXCR4 for cellular entry. In order to map the interaction of the FIV envelope surface unit (SU) with CXCR4, full-length FIV SU-Fc as well as constructs with deletions of extended loop L2, V3, V4, or V5 were produced in stable CHO cell lines. Binding studies were performed using these proteins on 3201 cells (CXCR4hi CD134−), with or without the CXCR4 inhibitor AMD3100. The findings established that SU binding to CXCR4 specifically requires the V3 region of SU. Synthetic peptides spanning the V3 region as well as a panel of monoclonal antibodies (MAbs) to SU were used to further map the site of CXCR4 interaction. Both the SU V3-specific antibodies and the full-length V3 peptide potently blocked binding of SU to CXCR4 and virus entry. By using a set of nested peptides overlapping a region of SU specifically recognized by CD134-dependent neutralizing V3 MAbs, we showed that the neutralizing epitope and the region required for CXCR4 binding are within the same contiguous nine-amino-acid sequence of V3. Site-directed mutagenesis was used to reveal that serine 393 and tryptophan 394 at the predicted tip of V3 are required to facilitate entry into the target cell via CXCR4. Although the amino acid sequences are not identical between FIV and HIV, the ability of FIV to bind and utilize both feline and human CXCR4 makes the feline model an attractive venue for development of broad-based entry antagonists.

Bioactive peptides in preventative healthcare: An overview of bioactivities and suggested methods to assess potential applications.

2021 ◽  
Vol 27 ◽  
Author(s):  
Maria Hayes
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bioactive Peptides ◽  
Infant Nutrition ◽  
Health Benefits ◽  
The United States ◽  
Amino Acid Sequences ◽  
Beneficial Effects ◽  
Potential Applications ◽  
Preventative Healthcare

: Food derived bioactive peptides can be generated from various protein sources and usually consist of between 2-30 amino acids with bulky, side-chain aromatic amino acids preferred in the ultimate and penultimate positions at the Cterminal end of the amino acid chain. They are reported to impart a myriad of preventative health beneficial effects to the consumer once ingested and these include heart health benefits through inhibition of enzymes including renin (EC 3.4.23.15) and angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1) within the renin angiotensin aldosterone system (RAAS); anti-inflammatory (due to inhibition of ACE-I and other enzymes) and anti-cancer benefits; prevention of type-2 diabetes through inhibition of dipeptidyl peptidase IV (DPP-IV), bone and dental strength, antimicrobial and immunomodulatory effects and several others. Peptides have also reported health benefits in the treatment of asthma, neuropathic pain, HIV and wound healing. However, the structure, amino acid composition and length of these peptides along with the quantity of peptide that can pass through the gastrointestinal tract and often the blood brain barrier (BBB) intact and reach the target organ is important for the realisation of these health effects in an in vivo setting. This paper aims to collate recent important research concerning the generation and detection of peptides in the laboratory. It discusses products currently available as preventative healthcare peptide options and relevant legislation barriers to place a food peptide product on the market. The review also highlights useful in silico computer based methods and analysis that may be used to generate specific peptide sequences from proteins whose amino acid sequences are known and also to determine if the peptides generated are unique and bioactive. The topic of food-derived bioactive peptides for health is of great interest to scientific research and industry due to evolving drivers in food product innovation including health and wellness for the elderly, infant nutrition and optimum nutrition for sports athletes as well as the humanisation of pets. This paper provides an overview of what is required to generate bioactive peptide containing hydrolysates; what methods should be used in order to characterise the health beneficial effects of these hydrolysates and the active peptide sequences, potential applications of bioactive peptides and legislative requirements in Europe and the United States. It also highlights success stories and barriers to the development of peptide containing food products that currently exist.

P1888Autoantiboides to Muscarinic M2 cholinoceptors in patients with paroxysmal atrial fibrillation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
E S Mironova ◽  
N A Mironova ◽  
N Y U Mironov ◽  
L Y U Layovich ◽  
S P Golitsyn ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Amino Acid ◽  
Healthy Subjects ◽  
Holter Monitoring ◽  
Active Role ◽  
Amino Acid Sequences ◽  
Healthy People ◽  
Extracellular Loop ◽  
Autoimmune Processes

Abstract Introduction Atrial fibrillation (AFib) is the most frequenly encountered arrhythmia. In the majority of cases AFib occurs in patients with cardiovascular diseases such as arterial hypertension (AH). In 10–15% of cases AFib occurs in the absense of comorbidities in structurally normal heart. It is reffered to as “lone AFib” and the cause of this arrhythmia remains unknown. Activation of cardiac M2-actylcholinoreceptors (M2-CR) leads to decrease in duration of atrial refractory periods that may contribute to development of AFib. Autoantibodies against M2-CR has cholinomimetic properties, but role of these autoantibodies in development and maintenance of AFib has not been studied. Purpose To assess autoantibodies against M2-CR in patients with paroxysmal lone AFib, in patients with AFib and AH and healthy people. Methods 100 patints with lone Afib, 100 patients with Afib and AH and 25 healthy people were included. Patients underwent clinical blood and urinlysis, assessment of biochemistry blood panel, 12-lead ECG, 24-hour Holter monitoring, echocardiography and stress-testing (treadmill or stress-echocardiography). Assesment of IgM and IgG autoantibodies to M2-CR was performed by indirect immunoenzyme assay. The following peptide molecules were used as epitopes for detection of autoantibodies: M1-amino acid sequence YTVIGYWPLGVVCDL (83–98) of the first extracellular loop of M2-CR; M2-sequence VRTVEDGECYIQFFSNAAVTFGTAI (168–192) of the second extracellular loop of M2-CR; M3-sequence NTFCAPCIPNTV (410–421) of the third extracellular loop of M2-CR, M4-short sequence VEDGECYIQFFS (171–182) of the second extracellular loop of M2-CR; M1+M4-chimeric molecule formed by sequences of the first and the second extacellular loops of M2-CR connected by disulfide bound YTVIGYWPLGVVCDL+VEDGECTIQFFS (83–98+171–182). Results IgG to M2-CR were found in 32% of patients with AFib and in 12% of healthy subjects (p<0,05). IgM to M2-CR were found in 25% of patients with AFib and in 32% of healthy subjects (p<0,05). In patients with lone AFib prevalence of IgG to M2-CR were greater than in patients with AFib and AH (39% vs 25%; p<0,05). Patients with lone AFib and AFib and AH had higher prevalence of IgG to all amino-acid sequences and difference was statistically significant for M2, M4, M1+M4 epitopes (see fig.). Prevalence of IgG to M2-CR epitopes Conclusion Higher prevalence of IgG to M2-CR in patients with AFib than in healthy subjects may suppose an active role of autoimmune processes in arrhythmogenesis.

scholarly journals Diverse CD81 Proteins Support Hepatitis C Virus Infection

2006 ◽  
Vol 80 (22) ◽  
pp. 11331-11342 ◽  
Author(s):  
Mike Flint ◽  
Thomas von Hahn ◽  
Jie Zhang ◽  
Michelle Farquhar ◽  
Christopher T. Jones ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Viral Entry ◽  
Hepatitis C Virus Infection ◽  
Extracellular Loop ◽  
Virus Attachment ◽  
Susceptibility To Infection ◽  
Cell Incubation ◽  
Large Extracellular Loop

ABSTRACT Hepatitis C virus (HCV) entry is dependent on CD81. To investigate whether the CD81 sequence is a determinant of HCV host range, we expressed a panel of diverse CD81 proteins and tested their ability to interact with HCV. CD81 large extracellular loop (LEL) sequences were expressed as recombinant proteins; the human and, to a low level, the African green monkey sequences bound soluble HCV E2 (sE2) and inhibited infection by retrovirus pseudotype particles bearing HCV glycoproteins (HCVpp). In contrast, mouse or rat CD81 proteins failed to bind sE2 or to inhibit HCVpp infection. However, CD81 proteins from all species, when expressed in HepG2 cells, conferred susceptibility to infection by HCVpp and cell culture-grown HCV to various levels, with the rat sequence being the least efficient. Recombinant human CD81 LEL inhibited HCVpp infectivity only if present during the virus-cell incubation, consistent with a role for CD81 after virus attachment. Amino acid changes that abrogate sE2 binding (I182F, N184Y, and F186S, alone or in combination) were introduced into human CD81. All three amino acid changes in human CD81 resulted in a molecule that still supported HCVpp infection, albeit with reduced efficiency. In summary, there is a remarkable plasticity in the range of CD81 sequences that can support HCV entry, suggesting that CD81 polymorphism may contribute to, but alone does not define, the HCV susceptibility of a species. In addition, the capacity to support viral entry is only partially reflected by assays measuring sE2 interaction with recombinant or full-length CD81 proteins.

scholarly journals Cell Surface Expression of Biologically Active Influenza C Virus HEF Glycoprotein Expressed from cDNA

1999 ◽  
Vol 73 (10) ◽  
pp. 8808-8812 ◽  
Author(s):  
Andrew Pekosz ◽  
Robert A. Lamb
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
Membrane Fusion ◽  
Cytoplasmic Tail ◽  
Surface Expression ◽  
Amino Acid Sequences ◽  
Biologically Active ◽  
Cell Surface Expression ◽  
Dependent Manner ◽  
Influenza C Virus

ABSTRACT The hemagglutinin, esterase, and fusion (HEF) glycoprotein of influenza C virus possesses receptor binding, receptor destroying, and membrane fusion activities. The HEF cDNAs from influenza C/Ann Arbor/1/50 (HEF-AA) and influenza C/Taylor/1223/47 (HEF-Tay) viruses were cloned and expressed, and transport of HEF to the cell surface was monitored by susceptibility to cleavage by exogenous trypsin, indirect immunofluorescence microscopy, and flow cytometry. Previously it has been found in studies with the C/Johannesburg/1/66 strain of influenza C virus (HEF-JHB) that transport of HEF to the cell surface is severely inhibited, and it is thought that the short cytoplasmic tail, Arg-Thr-Lys, is involved in blocking HEF cell surface expression (F. Oeffner, H.-D. Klenk, and G. Herrler, J. Gen. Virol. 80:363–369, 1999). As the cytoplasmic tail amino acid sequences of HEF-AA and HEF-Tay are identical to that of HEF-JHB, the data indicate that cell surface expression of HEF-AA and HEF-Tay is not inhibited by this amino acid sequence. Furthermore, the abundant cell surface transport of HEF-AA and HEF-Tay indicates that their cell surface expression does not require coexpression of another viral protein. The HEF-AA and HEF-Tay HEF glycoproteins bound human erythrocytes, promoted membrane fusion in a low-pH and trypsin-dependent manner, and displayed esterase activity, indicating that the HEF glycoprotein alone mediates all three known functions at the cell surface.

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