Structural Insights into Complexes of Glucose-Regulated Protein94 (Grp94) with Human Immunoglobulin G. Relevance for Grp94-IgG Complexes that Form In Vivo in Pathological Conditions

Abstract

While the mechanism by which Grp94 displays its chaperone function with client peptides in the cell has been elucidated extensively, much less is known about the nature and properties of how Grp94 can engage binding to proteins once it is exposed on the cell surface or liberated in the extra-cellular milieu, as occurs in pathological conditions. In this work, we wanted to investigate the molecular aspects and structural characteristics of complexes that Grp94 forms with human IgG, posing the attention on the influence that glycosylation of Grp94 might have on the binding capacity to IgG, and on the identification of sites involved in the binding. To this aim, we employed both native, fully glycosylated and partially glycosylated Grp94, and recombinant, non-glycosylated Grp94, as well as IgG subunits, in different experimental conditions, including the physiological setting of human plasma. Regardless of the species and type, Grp94 engages a similar, highly specific and stable binding with IgG that involves sites located in the N-terminal domain of Grp94 and the hinge region of whole IgG. Grp94 does not form stable complex with Fab, F(ab)2 or Fc. Glycosylation turns out to be an obstacle to the Grp94 binding to IgG, although this negative effect can be counteracted by ATP and spontaneously also disappears in time in a physiological setting of incubation. ATP does not affect at all the binding capacity of non-glycosylated Grp94. However, complexes that native, partially glycosylated Grp94 forms with IgG in the presence of ATP show strikingly different characteristics with respect to those formed in absence of ATP. Results have relevance for the mechanism regulating the formation of stable Grp94-IgG complexes in vivo, in the pathological conditions associated with the extra-cellular location of Grp94.

Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

“…To overcome the difficulty to identify the portion of the IgG heavy chain involved in binding Grp94 in vivo , we took the advantage of the analysis performed on complexes that Grp94 can also form in vitro with human IgG [ 9 ]. In previous works we demonstrated that these complexes have properties that partly overlap those displayed by native ones, being also characterized by an irreversible binding that confers on these complexes a particular resistance to tryptic digestion [ 11 ]. Taking into consideration these results and the finding that in diabetic plasma Grp94 is linked to IgG heavy chain ( Figure 2 ), we hypothesized that also in complexes formed in vitro the binding could occur in the same region of IgG.…”

“…Thus, by investigating in vitro formed Grp94-IgG complexes it was possible to make inference about the IgG region involved in Grp94 binding also in vivo . To this aim, mass analysis was conducted on heavy chain of human IgG incubated both alone and with recombinant rabbit Grp94, used as relevant substitute of human Grp94 to form nonimmune complexes [ 11 ]. Since in these experiments we used stoichiometric quantities of Grp94 and IgG, it was not necessary to submit samples to 2D-PAGE to separate IgG subunits that were easily evidenced by analyzing samples in reducing conditions in SDS-PAGE followed by Western blotting ( Figure 3(c) ).…”

“…Since complexes that Grp94 forms with IgG in vitro can reach big dimensions [ 11 ], we asked whether also complexes circulating in diabetic plasma could be visualized at the electronic microscopy. We thus analyzed IgG of both pooled and individual diabetic plasma and those of control subjects at the electronic microscopy.…”

“…In separate experiments, recombinant rabbit Grp94 was used to form complexes with human IgG, following the procedure detailed previously [ 11 ]. Briefly, Grp94 was incubated with IgG at the molar ratio of 1 : 2, and after incubation at 37°C for 2 h, the complex formation was evaluated in native PAGE by loading 5 μ g proteins onto 8.5% polyacrylamide gel, followed by Western blotting for detecting Grp94.…”

Co‐ and Post‐Translational Protein Folding in the ER

“…The other major ER‐resident chaperone of a general family is the Hsp90 Grp94, which caters to a more limited clientele , including Toll‐like receptors (TLRs), integrins , immunoglobulins , collagen , insulin‐like growth factors and members of the LDLR family . Although ATP binding and hydrolysis are essential steps for Grp94 chaperone activity , the exact mechanism of action of Grp94 has not been elucidated.…”