scholarly journals beta-Subunit 102-104 residues are crucial to confer FSH activity to equine LH/CG but are not sufficient to confer FSH activity to human CG

2001 ◽  
Vol 169 (1) ◽  
pp. 55-63 ◽  
Author(s):  
M Chopineau ◽  
N Martinat ◽  
C Galet ◽  
F Guillou ◽  
Y Combarnous
Keyword(s):  
Amino Acid ◽  
Activity Ratio ◽  
Structural Features ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Cos Cells ◽  
The Impact

Horse LH/CG (eLH/CG) and donkey LH/CG (dkLH/CG) are strictly LH-specific in their respective homologous species. However, both bind to the FSH receptors from non-equid species, whereas the zebra hormone (zbLH/CG) does not. The FSH/LH ratio of eLH/CG and of the alphadkbetae hybrid is about tenfold higher than that of dkLH/CG and of the alphaebetadk hybrid, showing that the betae subunit contains the structural features responsible for the high FSH activity of eLH/CG. Only six amino acid positions (51, 94, 95, 102, 103 and 106) are unique to the betae subunit when compared with the betadk and betazb subunits. The Gly-Pro and Val-Phe sequences in positions 102-103 of betadk and betae respectively were swapped by site-directed mutations and the mutated beta-subunits cDNAs were cotransfected in COS cells with either alphae or alphadk subunit cDNA. Other mutations were also introduced in 102-103 dkLH/CG beta-subunit: Ala-Ala, Gly-Ala or Ala-Pro. These mutations with Ala-Ala, Gly-Ala or Ala-Pro in the 102-103 betadkLH/CG subunit did not change the FSH/LH ratio of dkLH/CG but the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation promoted a marked increase in the FSH/LH activity ratio. This was observed with the two heterodimers containing alphae or alphadk. Conversely, the Val(102)-Phe(103) mutation in betae led to a dramatic drop in FSH/LH activity ratio of eLH/CG, to a level similar to that of dkLH/CG. Since all FSHs possess a Gly residue at position 104, we introduced the Gly(102)-Pro(103)-Arg(104)-->Val(102)-Phe(103)-Gly(104) mutation in betadk with the expectation that the increase in FSH activity observed with the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation could be potentiated. In fact, the additional Arg(104)-->Gly(104) mutation was found to abolish the increase in FSH activity observed with Gly(102)-Pro(103)-->Val(102)-Phe(103). Mutations Gly(102)-Pro(103)-->Val(102)-Arg(103) or Gly(102)-Pro(103)-Lys(104)--> Val(102)-Arg(103)-Gly(104) were also introduced in human CGbeta (hCGbeta) to compare the impact of these amino acid changes in the well-studied gonadotrophin hCG. The betahCG mutants obtained, co-expressed either with the human or the horse alpha-subunit, did not display any FSH activity. In conclusion, the 102-104 sequence in eLH/CG beta-subunits appears to be of utmost importance for their binding to FSH receptors. However, these results obtained with equid beta-subunits are not transposable to other gonadotrophins as similar mutations in hCGbeta did not lead to any increase in FSH activity.

scholarly journals Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.

1990 ◽  
Vol 172 (1) ◽  
pp. 335-345 ◽  
Author(s):  
A J Wardlaw ◽  
M L Hibbs ◽  
S A Stacker ◽  
T A Springer
Keyword(s):  
Amino Acid ◽  
Leukocyte Adhesion ◽  
Expression System ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Cdna Clones ◽  
Loss Of Function ◽  
Leukocyte Adhesion Deficiency ◽  
Mammalian Expression

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.

scholarly journals Mapping of antigenic and functional epitopes on the alpha- and beta-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1.

1983 ◽  
Vol 158 (2) ◽  
pp. 586-602 ◽  
Author(s):  
F Sanchez-Madrid ◽  
P Simon ◽  
S Thompson ◽  
T A Springer
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Features ◽  
Cell Interactions ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Complement Receptor ◽  
Receptor Function ◽  
Alpha Subunits ◽  
Sds Page

Mouse Mac-1, a complement receptor-associated surface structure on macrophages, and LFA-1, a function-associated structure on lymphocytes, comprise a novel family of leukocyte differentiation antigens participating in adhesive cell interactions. Mac-1 and LFA-1 contain alpha-subunits of 170,000 and 180,000 Mr, respectively, and beta-subunits of 95,000 Mr noncovalently associated in alpha 1 beta 1 complexes. The structural relation between the alpha- and between the beta-subunits, and the location of functionally important sites on the molecules, have been probed with antibodies. Both non-cross-reactive and cross-reactive monoclonal antibodies (MAb) and antisera prepared to the purified molecules or the LFA-1 alpha-subunits were used. Reactivity with individual subunits was studied by immunoprecipitation after dissociation induced by high pH treatment, or by immunoblotting after SDS-PAGE. Cross-reactive epitopes on Mac-1 and LFA-1 were found to be present on the beta-subunits, which were immunologically identical. Non-cross-reactive epitopes that are distinctive for Mac-1 or LFA-1 were localized to the alpha-subunits. MAb to LFA-1 alpha-subunit epitopes inhibited CTL-mediated killing. Two MAb to Mac-1 alpha-subunit epitopes but not a third MAb to a spatially distinct alpha-epitope inhibited complement receptor function. Neither function was inhibited by a MAb binding to a common beta-subunit epitope. Therefore, sites of Mac-1 and LFA-1 involved in their respective adhesion-related functions, as well as distinctive structural features, have been localized to the alpha-subunits.

scholarly journals Amino acid sequence of the human fibronectin receptor.

1987 ◽  
Vol 105 (3) ◽  
pp. 1183-1190 ◽  
Author(s):  
W S Argraves ◽  
S Suzuki ◽  
H Arai ◽  
K Thompson ◽  
M D Pierschbacher ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Calcium Binding ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Receptor Subunit ◽  
Fibronectin Receptor ◽  
Adhesion Receptor ◽  
Receptor Beta

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.

scholarly journals The substrate specificity of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 411-421 ◽  
Author(s):  
S J Yeaman ◽  
P Cohen ◽  
D C Watson ◽  
G H Dixon
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Kinase ◽  
Phosphorylation Site ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP-dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the alpha-subunit and of 14 amino acids around the phosphorylation site on the beta-subunit were shown to be: alpha-subunit Phe-Arg-Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly-Gly-His-Ser-Leu-Gly-Ala-Asp-Leu-Met-Ser-Pro-Ser-Phe-Leu-Ser-Pro-Gly-Thr-Ser-Val-Phe(Ser,Pro,Gly)His-Thr-Ser-Lys; beta-subunit, Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-VALIle-Tyr-Glu-Pro-Leu-Lys. The sites on histone H2B which are phosphorylated by cyclic AMP-dependent protein kinase in vitro were identified as serine-36 and serine-32. The amino acid sequence in this region is: Lys-Lys-Arg-Lys-Arg-Ser32(P)-Arg-Lys-Glu-Ser36(P)-Tyr-Ser-Val-Tyr-Val- [Iwai, K., Ishikawa, K. & Hayashi, H. (1970) Nature (London) 226, 1056-1058]. Serine-36 was phosphorylated at 50% of the rate at which the beta-subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7-fold more rapidly than was serine-32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N-terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.

scholarly journals Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry

1993 ◽  
Vol 294 (2) ◽  
pp. 357-363 ◽  
Author(s):  
R T Aplin ◽  
J E Baldwin ◽  
P L Roach ◽  
C V Robinson ◽  
C J Schofield
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Mass Spectrometry ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
British Library ◽  
Beta Subunits ◽  
Post Translational Modification ◽  
Acetic Acids

Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

Analysis of subunit assembly of the Na-K-ATPase

1994 ◽  
Vol 266 (3) ◽  
pp. C579-C589 ◽  
Author(s):  
D. M. Fambrough ◽  
M. V. Lemas ◽  
M. Hamrick ◽  
M. Emerick ◽  
K. J. Renaud ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sodium Pump ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Limited Region ◽  
Subunit Assembly ◽  
Alpha Subunits ◽  
Subunit Isoforms ◽  
P Type

The Na-K-ATPase, or sodium pump, is comprised of two subunits, alpha and beta. Each subunit spans the lipid bilayer of the cell membrane. This review summarizes our efforts to determine how the two subunits interact to form the functional ion transporter. Our major approach has been to observe the potential for subunit assembly when one or both subunits are truncated or present as chimeras that retain only a limited region of the Na-K-ATPase. DNAs encoding these altered subunit forms of the avian Na-K-ATPase are expressed in mammalian cells. Monoclonal antibodies specific for the avian beta-subunit are then used to purify newly synthesized avian beta-subunits, and the presence of accompanying alpha-subunits indicates that subunit assembly has occurred. The ectodomain of the beta-subunit (approximately residues 62-304) is sufficient for assembly with the alpha-subunit, and a COOH-terminal truncation of the beta-subunit that lacks aminoacyl residues beyond 162 will assemble inefficiently. A maximum of 26 aminoacyl residues of the alpha-subunit are necessary for robust assembly with the beta-subunit, when this sequence replaces the COOH-terminal half of the loop between membrane spans 7 and 8 in the SERCA1 Ca-ATPase. This region of the Ca-ATPase faces the lumen of the endoplasmic reticulum. These findings encourage study of other related questions, including whether there is preferential assembly of certain subunit isoforms and how various P-type ATPases are targeted to their appropriate subcellular compartments.

Context-Specific Function of the Engineered Peptide Domain of PHP.B

2021 ◽  
Author(s):  
R. Alexander Martino ◽  
Edwin C. Fluck ◽  
Jacqueline Murphy ◽  
Qiang Wang ◽  
Henry Hoff ◽  
...  
Keyword(s):  
Amino Acid ◽  
Blood Brain Barrier ◽  
Receptor Binding ◽  
Cell Types ◽  
Structural Features ◽  
Protein Domain ◽  
Brain Barrier ◽  
Target Cells ◽  
Blood Brain ◽  
The Impact

One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to 1) overcome poor transport through tissue barriers and 2) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert–capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood–brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B—including the 7-amino-acid loop and adjacent HVR VIII amino acids—can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood–brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. Importance Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors’ interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest: AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector’s performance.

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