TY  - JOUR
AB  - Immunoglobulins M (IgMs) are gaining increasing attention as biopharmaceuticals since their multivalent mode of binding can give rise to high avidity. Furthermore, IgMs are potent activators of the complement system. However, they are frequently difficult to express recombinantly and can suffer from low conformational stability. Here, the broadly neutralizing anti-HIV-1 antibody 2G12 was class-switched to IgM and then further engineered by introduction of 17 germline residues. The impact of these changes on the structure and conformational stability of the antibody was then assessed using a range of biophysical techniques. We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization. Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties. Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.
AU  - Chromikova, V.*
AU  - Mader, A.*
AU  - Hofbauer, S.*
AU  - Göbl, C.
AU  - Madl, T.
AU  - Gach, J.S.*
AU  - Bauernfried, S.*
AU  - Furtmüller, P.G.*
AU  - Forthal, D.N.*
AU  - Mach, L.*
AU  - Obinger, C.*
AU  - Kunert, R.*
C1  - 43715
C2  - 36798
SP  - 1536–1544
TI  - Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.
JO  - Biochim. Biophys. Acta-Proteins Proteom.
VL  - 1854
IS  - 10
PY  - 2015
SN  - 1570-9639
ER  - 

TY  - JOUR
AB  - The skeletal muscle is a metabolically active tissue that secretes various proteins. These so-called myokines have been proposed to affect muscle physiology and to exert systemic effects on other tissues and organs. Yet, changes in the secretory profile may participate in the pathophysiology of metabolic diseases. The present study aimed at characterizing the secretome of differentiated primary human skeletal muscle cells (hSkMC) derived from healthy, adult donors combining three different mass spectrometry based non-targeted approaches as well as one antibody based method. This led to the identification of 548 non-redundant proteins in conditioned media from hSkmc. For 501 proteins, significant mRNA expression could be demonstrated. Applying stringent consecutive filtering using SignalP, SecretomeP and ER_retention signal databases, 305 proteins were assigned as potential myokines of which 12 proteins containing a secretory signal peptide were not previously described. This comprehensive profiling study of the human skeletal muscle secretome expands our knowledge of the composition of the human myokinome and may contribute to our understanding of the role of myokines in multiple biological processes. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.
AU  - Hartwig, S.*
AU  - Raschke, S.*
AU  - Knebel, B.*
AU  - Scheler, M.
AU  - Irmler, M.
AU  - Passlack, W.*
AU  - Müller, S.*
AU  - Hanisch, F.-G.*
AU  - Franz, Z.*
AU  - Li, X.*
AU  - Dicken, H.D.*
AU  - Eckardt, K.*
AU  - Beckers, J.
AU  - Hrabě de Angelis, M.
AU  - Weigert, C.
AU  - Häring, H.-U.
AU  - Al-Hasani, H.*
AU  - Ouwens, D.M.*
AU  - Eckel, J.*
AU  - Kotzka, J.*
AU  - Lerch, S.*
C1  - 27181
C2  - 32568
CY  - Amsterdam
SP  - 1011-1017
TI  - Secretome profiling of primary human skeletal muscle cells.
JO  - Biochim. Biophys. Acta-Proteins Proteom.
VL  - 1844
IS  - 5
PB  - Elsevier Science
PY  - 2013
SN  - 1570-9639
ER  - 

TY  - JOUR
AB  - Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin 1. have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin 1. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR. Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by similar to 3 degrees C, both in T-50% and T-m. Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15D Delta C) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation. Removal of the C-terminal arm improved the thermal stability (T-m similar to+1.5 degrees C) of the truncated enzyme (VPR Delta C) as compared to the wild type VPR. Introduction of the N15D substitution into VPR Delta C improved the thermal stability further by about 3 degrees C, or to about the same extent as in the wild-type. However, contrary to what was observed for the wild type, the introduction of the putative salt bridge did not affect the catalytic properties (k(cat)) of the C-terminal truncated enzyme.
AU  - Sigurdardottir, A.G.*
AU  - Arnórsdóttir, J.*
AU  - Thorbjarnardóttir, S.H.*
AU  - Eggertsson, G.*
AU  - Suhre, K.
AU  - Kristjánsson, M.M.*
C1  - 1659
C2  - 26777
SP  - 512-518
TI  - Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.
JO  - Biochim. Biophys. Acta-Proteins Proteom.
VL  - 1794
IS  - 3
PB  - Elsevier Science Bv
PY  - 2009
SN  - 1570-9639
ER  -