Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor

Abstract

IntroductionWe recently identi®ed the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP) (Rieger et al., 1997; for reviews see Rieger et al., 1999;Gauczynski et al., 2001a). Employing a series of neuronal and non-neuronal cells, we proved that the 37-kDa LRP/67-kDa high-af®nity laminin receptor (LR) acts as the receptor for the cellular PrP (Gauczynski et al., 2001b). In the present manuscript we used the yeast twohybrid system and cell-binding studies on neuronal as well as non-neuronal cells involving the Semliki Forest virus (SFV) system (for reviews see Liljestrom and Garoff, 1991;Tubulekas et al., 1997) to identify domains on the PrP and the LRP involved in the PrP±LRP interaction on the cell surface. We identi®ed two binding domains for LRP on PrP termed PrPLRPbd1 and PrPLRPbd2. The ®rst one binds directly to LRP, whereas the second one depends on the presence of heparan sulfate proteoglycans (HSPGs) on the cell surface. The yeast two-hybrid system and cell-binding assays on wild-type and mutant HSPGde®cient Chinese hamster ovary (CHO) cells also identi®ed two binding domains for PrP on LRP.The relationship between 37-kDa LRP and 67-kDa LR is not yet fully understood and has been explained with homodimerization of 37-kDa LRP (Landowski et al., 1995) or an additional factor, such as a polypeptide (Castronovo et al., 1991), which might bind to 37-kDa LRP to form the 67-kDa form of the receptor. The 67-kDa heterodimer might be stabilized by hydrophobic interactions mediated by fatty acids such as palmitate, oleate and stearate bound to 37-kDa LRP and to a galectin-3 (gal-3) cross reacting polypeptide (Landowski et al., 1995;Buto et al., 1998). However, we recently proved that the b-galactoside lectin gal-3 is not present on the surface of neuronal or non-neuronal cells used for PrP-binding/ internalization studies (Gauczynski et al., 2001b) and antigal-3 antibodies failed to compete for the 37-kDa LRP/67-kDa LR-mediated binding and internalization of the cellular PrP (Gauczynski et al., 2001b), suggesting that gal-3 is not a partner of the 37-kDa LRP in this context. In this study we investigated by a yeast two-hybrid system analysis whether gal-3 interacts with 37-kDa LRP and/or the cellular PrP. In addition, we investigated whether 37-kDa LRP interacts with itself in the yeast two-hybrid and analysed the monomer/dimer status of the receptor by sizeexclusion chromatography. Both PrP (Gabizon et al., 1993;Caughey et al., 1994;Chen,S.G. et al., 1995;Brimacombe et al., 1999) and the 37-kDa/67-kDa LR (Guo et al., 1992;Kazmin et al., 2000) bind to heparan sulfates. HSPGs are required for the binding of the ®broblast growth factor (FGF) to its FGFR receptor (Yayon et al., 1991;Spivak et al., 1994;Venkataraman et al., 1999) and act as initial attachment receptors for bacteria (Chen,T. et al., 1995) and viruses including alphaviruses (Byrnes and Grif®n, 1998), human immunode®ciency virus (HIV) type 1 (Mondor et al., 1998) and vaccinia virus (Chung et al., 1998). Heparan sulfates are...

Inter- and Intra-octarepeat Cu(II) Site Geometries in the Prion Protein

“…By contrast, when the two dipeptides form part of the octarepeat units of different molecules, the inter-repeat site is intermolecular, as in Cu(II)⅐␣BoPrP-(24 -242) polymers formed using protein concentrations above 40 M (28). To envisage the interrepeat Cu(II) as a dimer of His-Gly dipeptides is also compatible with the observed retention of ␣PrP in a Cu(II)-loaded matrix (9,26) and the formation of a hetero-coordination complex with sulfated glycosaminoglycans and their assemblies (25,27).…”

“…In the presence of Cu(II), PrP (as PrP C or ␣PrP) is retained in inner membrane immobilized metal ion affinity chromatography matrices, binds tighter to sulfated glycosaminoglycans and their complexes, and forms homo-polymers at protein concentrations above 40 M (9,(25)(26)(27)(28). Despite the large number of studies on Cu(II)-PrP interaction, the structural basis of the Cu(II)-mediated ␣PrP assembly and of the binding cooperativity are still unknown.…”

Prion and doppel proteins bind to granule cells of the cerebellum

“…1 A). At the N terminus, we found that HuM-E123, which recognizes the region of MoPrP (30)(31)(32)(33)(34)(35)(36)(37), bound weakly, and that HuM-E149, a HuM-Fab specific to the octarepeat region, bound even more weakly than HuM-E123 (Fig. 1 A).…”

“…These findings raise the possibility that the cells of the molecular layer express an auxiliary protein or proteins involved in prion formation, which is provisionally designated protein X and is likely to be distinct from a protein that may mediate Dpl-induced degeneration. The search for protein X has been frustrating because many proteins are known to bind to PrP C , but none have been shown to participate in PrP Sc formation (17,18,(28)(29)(30)(31)(32)(33)(34)(35)(36). Although the binding of the dominant-negative MoPrP(Q218K)-Fc to cells in the molecular layer where PrP C is expressed builds a case for the possibility that MoPrP-(Q218K)-Fc is binding to protein X, the absence of PrP Sc deposition in the molecular layer requires us to hypothesize that PrP Sc formed in molecular layer is readily transported to the cerebellar white matter where PrP Sc is found.…”

The use of non-prion biomarkers for the diagnosis of Transmissible Spongiform Encephalopathies in the live animal

“…Studies [34,92] have indicated that LR/ LRP may have a role in susceptibility to oral TSE infection, but there is no substantial evidence to suggest that they are surrogate markers of TSE diseases. Other studies have shown specific interactions between PrP and LRP [32,48]. Boehringer Ingelheim reports to have developed antibodies and oligonucleotides specific to the marker proteins and their mRNAs.…”