Peptide Binding Inhibits Aggregation of Soluble MHC Class II in Solution

Abstract

Affinity-purified major histocompatability complex (MHC) class II molecules are known to bind antigenic peptide in vitro. This peptide-bound MHC class II is known to undergo a change in structure upon stable binding of antigenic peptide. Previous results from our, and other laboratories, have suggested a relationship between MHC class II structure and peptide association that enables class II to enter into a stable conformation upon peptide binding. In this report we describe that stable binding of high-affinity antigenic peptide to MHC class II molecule results in transition of aggregated purified MHC class II proteins to a stable heterodimeric state. Such transition was demonstrated by using purified human HLA-DR2 class II molecule and high-affinity myelin basic protein (MBP) 83-102)Y83 peptide. Highly aggregated purified DR2 (high molecular weight; HMW) was first separated from heterodimer (low molecular weight: LMW) in the presence of 50-fold molar excess of MBP(83-102)Y83 peptide. We then show that the aggregated HMW preparation can be successfully converted into a stable dimer by further incubation with MBP(83-102)Y83 and changing various binding parameters such as pH, temperature, reducing agent, and peptide concentrations. Under optimized conditions, the highly aggregated inactive DR2 molecules can be completely loaded with the antigenic peptide. The transformed heterodimers with bound peptide prepared by this method are biologically active, as shown by their ability to induce the production of gamma-interferon by SS8T-transformed human T cells. These results suggest that in solution, MHC class II molecules may be aggregated in the absence of bound peptide. Such aggregated MHC class II molecules can be converted to stable and biologically active heterodimers in the presence of high-affinity antigenic peptide.

Use of MHC class II tetramers to investigate CD4+ T cell responses: Problems and solutions

“…In Figure 1b, an SDS stability assay shows that upon loading of HLA‐DR*1101 molecules with the TT derived promiscuous HLA‐class II peptide p2, about 50% of the heterodimer is stabilized in the presence of 0.2% of SDS at room temperature. Moreover, empty MHC class II molecules are less stable and tend to aggregate (8, 19). To reduce the instability of empty MHC class II upon production and purification, while maintaining the flexibility of the à la carte peptide loading, soluble recombinant HLA‐DR molecules have been produced with the nonantigenic invariant chain (Ii)‐derived CLIP peptide, which is naturally incorporated into the MHC class II molecule groove during their biosynthesis.…”

Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide

“…Phage display of MHC has proved to be particularly challenging, and only three examples of successful display of class I MHC have been demonstrated to date (Kurokawa et al, 2002;Le Doussal et al, 2000;Vest Hansen et al, 2001). The lack of MHC phage display data is unsurprising as these proteins are natively heterodimeric with intrachain disulfide bonds; furthermore, at least some MHC proteins demonstrate poor stability or nonnative conformations in the absence of bound antigen peptide (Arimilli et al, 1999;Germain and Rinker, 1993). These issues complicate heterologous expression in prokaryotic hosts, which is a prerequisite for phage display.…”

Σχεδιασμός Σύνθεση Και Αξιολόγηση Αναλόγων Των Ανοσοκυρίαρχων Επιτόπων Των Προτεϊνών Της Μυελίνης Που Εμπλέκονται Στη Σκλήρυνση Κατά Πλάκας

“…Σχήμα 5: Η πρωτεΐνη HLA II κωδικοποιείται στο χρωμόσωμα 6, είναι μία διαμεμβρανική ετεροδιμερής πρωτεΐνη μοριακού βάρους 60 kDa, η οποία αποτελείται από την α πεπτιδική αλυσίδα με μοριακό βάρος 32 kDa και από τη β αλυσίδα με μοριακό βάρος 28 kDa.Οι αλυσίδες α και β είναι δομικά ομόλογες (Arimilli et al 1999;Ladiabetes 2011;Winslow 2012). (Polman et al 2006).…”