scholarly journals Effect of Trastuzumab–HER2 Complex Formation on Stress-Induced Modifications in the CDRs of Trastuzumab

2022 ◽  
Vol 9 ◽  
Author(s):  
Baubek Spanov ◽  
Victoria Aboagye ◽  
Oladapo Olaleye ◽  
Natalia Govorukhina ◽  
Nico C. van de Merbel ◽  
...  
Keyword(s):  
Complex Formation ◽  
Size Exclusion ◽  
Major Influence ◽  
Target Antigen ◽  
Moderate Degree ◽  
Free Antibody ◽  
Exclusion Chromatography ◽  
Complementarity Determining Regions ◽  
C Complex ◽  
Acid Isomerization

Asparagine deamidation and aspartic acid isomerization in the complementarity determining regions (CDRs) of monoclonal antibodies may alter their affinity to the target antigen. Trastuzumab has two hot spots for deamidation and one position for isomerization in the CDRs. Little is known how complex formation with its target antigen HER2 affects these modifications. Modifications in the CDRs of trastuzumab were thus compared between the free antibody and the trastuzumab–HER2 complex when stressed under physiological conditions at 37°C. Complex formation and stability of the complex upon stressing were assessed by size-exclusion chromatography. Deamidation of light-chain Asn-30 (Lc-Asn-30) was extensive when trastuzumab was stressed free but reduced about 10-fold when the antibody was stressed in complex with HER2. Almost no deamidation of heavy-chain (Hc-Asn-55) was detected in the trastuzumab–HER2 complex, while deamidation was observed when the antibody was stressed alone. Hc-Asp-102 isomerization, a modification that critically affects biological activity, was observed to a moderate degree when the free antibody was stressed but was not detected at all in the trastuzumab–HER2 complex. This shows that complex formation has a major influence on critical modifications in the CDRs of trastuzumab.

In vitro digestibility study of thermal oxidized palm oleins Estudio de la digestibilidad in vitro de oleínas de palma termooxidadas

2000 ◽  
Vol 6 (6) ◽  
pp. 449-456 ◽  
Author(s):  
F.J. Sánchez-Muniz ◽  
R. Arroyo ◽  
J.M. Sánchez-Montero ◽  
C. Cuesta
Keyword(s):  
Pancreatic Lipase ◽  
High Performance ◽  
Palm Olein ◽  
Size Exclusion ◽  
In Vitro Digestibility ◽  
Moderate Degree ◽  
Before And After ◽  
Exclusion Chromatography ◽  
Hydrolysis Of

Information on digestibility and absorption of oils and fats used for frying is under debate. To get knowledge on this, unused palm olein (9.27 ± 0.10% w/w polar content), used frying palm olein with a moderate degree of alteration (14.81 ± 0.90% w/w polar content) and highly altered used frying palm olein (26.36 ± 0.30% w/w polar content) and their respective nonpolar and polar fractions were studied. Samples were analyzed by high-performance size-exclusion chromatography before and after a 20-min in vitro incubation with pancreatic lipase. Formation of monoacylglycerols and free fatty acids reflected no relevant differences between unused and moderately altered oleins, whereas the most altered olein was hydrolyzed to a much lesser degree. The presence of oligomers (dimers and polymers of triacylglycerols) negatively affected the hydrolysis of triacylglycerol monomers in whole oleins. The hydrolysis of these monomers in the isolated nonpolar and polar fractions ranked between 60.2% and 78.5%. Oligomers were efficiently hydrolyzed by pancreatic lipase in whole un used and moderately altered oleins but not in the most altered one. Polymers from isolated polar fractions were poorly hydrolyzed or not hydrolyzed at all. These data suggest that whole oleins contained some compounds that increase susceptibility of oligomers to enzymatic hydrolysis and that such compounds were not present in the polar fraction.

scholarly journals An N-terminally truncated form of cyclic GMP–dependent protein kinase Iα (PKG Iα) is monomeric and autoinhibited and provides a model for activation

2018 ◽  
Vol 293 (21) ◽  
pp. 7916-7929 ◽  
Author(s):  
Thomas M. Moon ◽  
Jessica L. Sheehe ◽  
Praveena Nukareddy ◽  
Lydia W. Nausch ◽  
Jessica Wohlfahrt ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Muscle Relaxation ◽  
Size Exclusion ◽  
Smooth Muscle Relaxation ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Exclusion Chromatography ◽  
Dependent Protein ◽  
C Complex ◽  
Cooperative Activation

The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMP-dependent protein kinase (PKA). Here, we examined PKG Iα activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1–53 (Δ53). We found that the construct exists as a monomer as assessed by whole-protein MS, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that Δ53 has a similar shape to the heterodimeric RIα–C complex of PKA. Moreover, we found that the Δ53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length PKG Iα as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the Δ53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Iα activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers.

Phospholipid-Protein Interactions in the Formation of Prothrombin Activator

1965 ◽  
Vol 14 (03/04) ◽  
pp. 431-444 ◽  
Author(s):  
E. R Cole ◽  
J. L Koppel ◽  
J. H Olwin
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Calcium Ions ◽  
Fixed Number ◽  
Factor V ◽  
Clotting Factors ◽  
Number Of Binding Sites ◽  
C Complex ◽  
Autoprothrombin C

SummarySince Ac-globulin (factor V) is involved in the formation of prothrombin activator, its ability to complex with phospholipids was studied. Purified bovine Ac-globulin was complexed to asolectin, there being presumably a fixed number of binding sites on the phospholipid micelle for Ac-globulin. In contrast to the requirement for calcium ions in the formation of complexes between asolectin and autoprothrombin C, calcium ions were not required for complex formation between asolectin and Ac-globulin to occur ; in fact, the presence of calcium prevented complex formation occurring, the degree of inhibition being dependent on the calcium concentration. By treating isolated, pre-formed aso- lectin-Ac-globulin complexes with calcium chloride solutions, Ac-globulin could be recovered in a much higher state of purity and essentially free of asolectin.Complete activators were formed by first preparing the asolectin-calcium- autoprothrombin C complex and then reacting the complex with Ac-globulin. A small amount of this product was very effective as an activator of purified prothrombin without further addition of calcium or any other cofactor. If the autoprothrombin C preparation used to prepare the complex was free of traces of prothrombin, the complete activator was stable for several hours at room temperature. Stable preparations of the complete activator were centrifuged, resulting in the sedimentation of most of the activity. Experimental evidence also indicated that activator activity was highest when autoprothrombin C and Ac-globulin were complexed to the same phospholipid micelle, rather than when the two clotting factors were complexed to separate micelles. These data suggested that the in vivo prothrombin activator may be a sedimentable complex composed of a thromboplastic enzyme, calcium, Ac-globulin and phospholipid.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

Sign in / Sign up

Export Citation Format

Share Document