Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Protein Sci ; 30(2): 485-496, 2021 02.
Article in English | MEDLINE | ID: mdl-33277949

ABSTRACT

The insulin epitopes for two monoclonal antibodies (mAbs), OXI-005 and HUI-018, commonly used in combination for insulin concentration determination in sandwich assays, were determined using X-ray crystallography. The crystal structure of the HUI-018 Fab in complex with human insulin (HI) was determined and OXI-005 Fab crystal structures were determined in complex with HI and porcine insulin (PI) as well as on its own. The OXI-005 epitope comprises insulin residues 1,3,4,19-21 (A-chain) and 25-30 (B-chain) and for HUI-018 residues 7,8,10-14,17 (A-chain) and 5-7, 10, 14 (B-chain). The areas of insulin involved in interactions with the mAb are 20% (OXI-005) and 24% (HUI-018) of the total insulin surface. Based on the Fab complex crystal structures with the insulins a molecular model for simultaneous binding of the Fabs to PI was built and this model was validated by small angle X-ray scattering measurements for the ternary complex. The epitopes for the mAbs on insulin were found well separated from each other as expected from luminiscent oxygen channeling immunoassay results for different insulins (HI, PI, bovine insulin, DesB30 HI, insulin glargine, insulin lispro). The affinities of the OXI-005 and HUI-018 Fabs for HI, PI, and DesB30 HI were determined using surface plasmon resonance. The KD s were found to be in the range of 1-4 nM for the HUI-018 Fab, while more different for the OXI-005 Fab (50 nM for HI, 20 nM for PI and 400 nM for DesB30 HI) supporting the importance of residue B30 for binding to OXI-005.


Subject(s)
Antibodies, Monoclonal/chemistry , Epitopes/chemistry , Immunoglobulin Fab Fragments/chemistry , Insulin/chemistry , Models, Molecular , Crystallography, X-Ray , Epitope Mapping , Humans
2.
Org Biomol Chem ; 18(25): 4717-4722, 2020 07 01.
Article in English | MEDLINE | ID: mdl-32525502

ABSTRACT

Methods for chemical modification of native proteins in a controlled fashion are in high demand. Here, a novel protocol that exploits bifunctional reagents for transient targeting of solvent exposed disulphides to direct the introduction of a single exogenous reactive thiol handle at a lysine side chain has been developed. The protocol has successfully been applied to functionalize six different Fabs and human growth hormone.


Subject(s)
Disulfides/chemistry , Growth Hormone/chemistry , Humans , Lysine/chemistry , Molecular Structure
3.
Biochemistry ; 59(14): 1410-1419, 2020 04 14.
Article in English | MEDLINE | ID: mdl-32208682

ABSTRACT

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding. Isothermal titration calorimetry (ITC) binding profiles showed high-affinity binding (∼100-1000 nM) of one somapacitan molecule and low-affinity binding (∼1000-10000 nM) of one to two somapacitan molecules to HSA. The high-affinity site was identified in HSA domain III using size exclusion chromatography (SEC) and ITC. SEC studies showed that the neonatal Fc receptor shields one binding site for somapacitan, indicating its position in domain III. A crystal structure of somapacitan in complex with HSA optimized for neonatal Fc receptor binding, having four amino acid residue replacements, identified a low-affinity site in fatty acid-binding site 6 (domain II). Surface plasmon resonance (SPR) showed these replacements affect the kinetics of the high-affinity binding site. Furthermore, small-angle X-ray scattering and SPR brace two somapacitan-binding sites on HSA.


Subject(s)
Growth Hormone/chemistry , Serum Albumin, Human/chemistry , Binding Sites , Growth Hormone/analogs & derivatives , Growth Hormone/metabolism , Humans , Kinetics , Protein Binding , Protein Domains , Serum Albumin, Human/metabolism , Surface Plasmon Resonance
4.
Biochemistry ; 58(24): 2750-2759, 2019 06 18.
Article in English | MEDLINE | ID: mdl-31117388

ABSTRACT

Aggregation can be a major challenge in the development of antibody-based pharmaceuticals as it can compromise the quality of the product during bioprocessing, formulation, and drug administration. To avoid aggregation, developability assessment is often run in parallel with functional optimization in the early screening phases to flag and deselect problematic molecules. As developability assessment can be demanding with regard to time and resources, there is a high focus on the development of molecule design strategies for engineering molecules with a high developability potential. Previously, Dudgeon et al. [(2012) Proc. Natl. Acad. Sci. U. S. A. 109, 10879-10884] demonstrated how Asp substitutions at specific positions in human variable domains and single-chain variable fragments could decrease the aggregation propensity. Here, we have investigated whether these Asp substitutions would improve the developability potential of a murine antigen binding fragment (Fab). A full combinatorial library consisting of 393 Fab variants with single, double, and triple Asp substitutions was first screened in silico with Rosetta; thereafter, 26 variants with the highest predicted thermodynamic stability were selected for production. All variants were subjected to a set of developability studies. Interestingly, most variants had thermodynamic stability on par with or improved relative to that of the wild type. Twenty-five of the variants exhibited improved nonspecificity. Half of the variants exhibited improved aggregation resistance. Strikingly, while we observed remarkable improvement in the developability potential, the Asp substitutions had no substantial effect on the antigenic binding affinity. Altogether, by combining the insertion of negative charges and the in silico screen based on computational models, we were able to improve the developability of the Fab rapidly.


Subject(s)
Aspartic Acid/chemistry , Immunoglobulin Fab Fragments/chemistry , Amino Acid Substitution , Animals , Antigens/immunology , Computer Simulation , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Mice , Peptide Library , Protein Multimerization/genetics , Protein Stability
5.
Bioconjug Chem ; 29(9): 3129-3143, 2018 09 19.
Article in English | MEDLINE | ID: mdl-30168709

ABSTRACT

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.


Subject(s)
Albumins/metabolism , Human Growth Hormone/metabolism , Alkylation , Animals , Area Under Curve , Half-Life , Oxidation-Reduction , Protein Binding , Rats , Rats, Sprague-Dawley
6.
Anal Chem ; 87(12): 5973-80, 2015 Jun 16.
Article in English | MEDLINE | ID: mdl-25978680

ABSTRACT

Human growth hormone (hGH), and its receptor interaction, is essential for cell growth. To stabilize a flexible loop between helices 3 and 4, while retaining affinity for the hGH receptor, we have engineered a new hGH variant (Q84C/Y143C). Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map the impact of the new disulfide bond on the conformational dynamics of this new hGH variant. Compared to wild type hGH, the variant exhibits reduced loop dynamics, indicating a stabilizing effect of the introduced disulfide bond. Furthermore, the disulfide bond exhibits longer ranging effects, stabilizing a short α-helix quite distant from the mutation sites, but also rendering a part of the α-helical hGH core slightly more dynamic. In the regions where the hGH variant exhibits a different deuterium uptake than the wild type protein, electron transfer dissociation (ETD) fragmentation has been used to pinpoint the residues responsible for the observed differences (HDX-ETD). Finally, by use of surface plasmon resonance (SPR) measurements, we show that the new disulfide bond does not compromise receptor affinity. Our work highlight the analytical potential of HDX-ETD combined with functional assays to guide protein engineering.


Subject(s)
Disulfides/chemistry , Human Growth Hormone/chemistry , Protein Engineering , Deuterium Exchange Measurement , Electron Transport , Humans , Mass Spectrometry , Models, Molecular , Protein Conformation
7.
Eur J Pharm Sci ; 40(4): 273-81, 2010 Jul 11.
Article in English | MEDLINE | ID: mdl-20380877

ABSTRACT

In the pharmaceutical industry, protein drugs are modified by, for instance, glycosylation in order to obtain protein drugs with improved delivery profiles and/or increased stability. The effect of glycosylation on protein adsorption behaviour is one of the stability aspects that must be evaluated during development of glycosylated protein drug products. We have studied the effect of glycosylation on the adsorption behaviour of Thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces using total internal reflection fluorescence, surface plasmon resonance, far-UV circular dichroism and fluorescence. Three glyco-variants were used, namely the mono-glycosylated wildtype T. lanuginosus lipase, a non-glycosylated variant and a penta-glycosylated variant, the latter two containing one and nine amino acid substitutions, respectively. All the glycosylations were N-linked and contained no charged sugar residues. Glycosylation did not affect the adsorption of wildtype T. lanuginosus lipase to the hydrophobic surfaces. The number of molecules adsorbing per unit surface area, the structural changes occurring upon adsorption, and the orientation upon adsorption were found to be unaffected by the varying glycosylation. However, the interaction with a hydrophilic surface was different between the three glyco-variants. The penta-glycosylated T. lanuginosus lipase adsorbed, in contrast to the two other glyco-variants. In conclusion, adsorption of T. lanuginosus lipase to hydrophobic surfaces was not affected by N-linked glycosylation. Only penta-glycosylated T. lanuginosus lipase adsorbed to the hydrophilic surface, apparently due to its increased net charge of +3 caused by amino acid substitutions in the primary sequence.


Subject(s)
Ascomycota/enzymology , Enzymes, Immobilized/chemistry , Fungal Proteins/chemistry , Lipase/chemistry , Adsorption , Amino Acid Substitution , Circular Dichroism , Enzyme Stability , Glycosylation , Hydrophobic and Hydrophilic Interactions , Kinetics , Molecular Weight , Mutant Proteins/chemistry , Organosilicon Compounds/chemistry , Protein Structure, Secondary , Quartz/chemistry , Recombinant Proteins/chemistry , Silanes/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Surface Plasmon Resonance , Surface Properties
8.
Stem Cells Dev ; 16(6): 965-78, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18047418

ABSTRACT

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to differentiate into various different cell lineages and can generate bone, cartilage and adipose tissue. MSCs are presently characterized using a broad range of different cell-surface markers that are not exclusive to MSCs and not sensitive to culture conditions or differentiation capacity. We show that the integrin subunits alpha10 and alpha11 of the collagen binding integrins alpha10beta1 and alpha11beta1 are expressed by human MSCs in monolayer cultures. We also demonstrate that the expression of alpha10 increases, while alpha1 and alpha11 decrease, during aggregate culture of MSCs in chondrogenic medium. Alpha10beta1 is expressed by chondrocytes in cartilage, whereas alpha11beta1 integrins are predominantly expressed by subsets of the fibroblastic lineage. In extensive monolayer cultures of MSCs, alpha10 expression is down-regulated. We show that this down-regulation is reversed by fibroblast growth factor-2 (FGF-2) treatment. Addition of FGF-2 to MSCs not only results in increased alpha10 expression, but also in decreased alpha11 expression. FGF-2 treatment of MSCs has been shown to keep the cells more multipotent and also induces cell proliferation and Sox-9 up-regulation. We demonstrate improved chondrogenecity as well as increased collagen-dependant migratory potential of FGF-2-treated MSCs having a high alpha10 expression. We also demonstrate expression of alpha10 and alpha11 integrin subunits in the endosteum and periosteum of mice, but very low or not detectable expression levels in freshly aspired human or mouse BM. We show that MSCs with high chondrogenic differentiation potential are highly alpha10 positive and propose alpha10 as a potential marker to predict the differentiation state of MSCs.


Subject(s)
Cell Differentiation/physiology , Chondrocytes/cytology , Fibroblast Growth Factor 2/pharmacology , Integrin alpha Chains/genetics , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/physiology , Cell Movement , Collagen/physiology , Flow Cytometry , Gene Expression Regulation/drug effects , Growth Substances/pharmacology , Humans , Mesenchymal Stem Cells/drug effects , Polymerase Chain Reaction , Up-Regulation
9.
J Biol Chem ; 278(39): 37698-704, 2003 Sep 26.
Article in English | MEDLINE | ID: mdl-12840020

ABSTRACT

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.


Subject(s)
Collagen Type II/chemistry , Collagen Type VI/chemistry , Extracellular Matrix Proteins/chemistry , Glycoproteins/chemistry , Microfibrils/chemistry , Proteoglycans/chemistry , Aggrecans , Animals , Biglycan , Cells, Cultured , Chondrocytes/chemistry , Decorin , Extracellular Matrix/chemistry , Lectins, C-Type , Leucine-Rich Repeat Proteins , Matrilin Proteins , Microscopy, Electron , Proteins/chemistry , Rats
10.
J Biol Chem ; 277(51): 49120-6, 2002 Dec 20.
Article in English | MEDLINE | ID: mdl-12354766

ABSTRACT

The ability of the leucine-rich repeat (LRR) proteins biglycan, decorin, and chondroadherin to interact with collagen VI and influence its assembly to supramolecular structures was studied by electron microscopy and surface plasmon resonance measurements in the BIAcore 2000 system. Biglycan showed a unique ability to organize collagen VI into extensive hexagonal-like networks over a time period of only a few minutes. Only the intact molecule, substituted with two dermatan sulfate chains, had this capacity. Intact decorin, with one dermatan sulfate chain only, was considerably less efficient, and aggregates of organized collagen VI were found only after several hours. Chondroadherin without glycosaminoglycan substitutions did not induce any ordered collagen VI organization. However, all three related LRR proteins were shown to interact with collagen VI using electron microscopy and surface plasmon resonance. Biglycan and decorin were exclusively found close to the N-terminal parts of the collagen VI tetramers, whereas chondroadherin was shown to bind close to both the N- and C-terminal parts of collagen VI. In the formed hexagonal networks, biglycan was localized to the intra-network junctions of the collagen VI filaments. This was demonstrated by electron microscopy after negative staining of gold-labeled biglycan in aggregation experiments with collagen VI.


Subject(s)
Collagen Type VI/chemistry , Collagen Type VI/metabolism , Proteoglycans/physiology , Animals , Biglycan , Cattle , Cells, Cultured , Chondroitin ABC Lyase/metabolism , DNA, Complementary/metabolism , Dermatan Sulfate/metabolism , Dimerization , Dose-Response Relationship, Drug , Extracellular Matrix Proteins/metabolism , Glycosaminoglycans/chemistry , Humans , Microscopy, Electron , Placenta/pathology , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Proteoglycans/metabolism , Recombinant Proteins/metabolism , Surface Plasmon Resonance , Temperature , Time Factors , Tissue Distribution
11.
Biochem J ; 367(Pt 3): 715-21, 2002 Nov 01.
Article in English | MEDLINE | ID: mdl-12180907

ABSTRACT

Matrilin-2 is a component of extracellular filamentous networks. To study the interactions by which it can be integrated into such assemblies, full-length and truncated forms of matrilin-2 were recombinantly expressed in HEK-293 cells and purified from conditioned medium. The recombinant proteins, when used in interaction assays, showed affinity to matrilin-2 itself, but also to other collagenous and non-collagenous extracellular matrix proteins. The interaction between matrilin-2 and collagen I was studied in greater detail and could be shown to occur at distinct sites on the collagen I molecule and to have a K (D) of about 3 x 10(-8) M. Interactions with some non-collagenous protein ligands were even stronger, with matrilin-2 binding to fibrillin-2, fibronectin and laminin-1-nidogen-1 complexes, with K (D) values in the range of 10(-8)-10(-11) M. Co-localization of matrilin-2 with these ligands in the dermal-epidermal basement membrane, in the microfibrils extending from the basement membrane into the dermis, and in the dermal extracellular matrix, indicates a physiological relevance of the interactions in the assembly of supramolecular extracellular matrix structures.


Subject(s)
Extracellular Matrix Proteins/metabolism , Glycoproteins/metabolism , Base Sequence , Cell Line , Collagen Type I/metabolism , DNA Primers , Humans , Ligands , Matrilin Proteins , Microscopy, Electron , Protein Binding , Recombinant Proteins/metabolism , Skin/metabolism , Surface Plasmon Resonance
SELECTION OF CITATIONS
SEARCH DETAIL
...