Proteolytic processing and glycosylation influence formation of porcine prion protein complexes

Abstract

High level of heterogeneity seems to be a ubiquitous feature of mammalian PrPs (prion proteins) and may be relevant to the pathogenesis of prion diseases. In the present study, we describe the heterogeneity of PrP(C) (cellular form of PrP) from porcine brain. It was disclosed and characterized by a combination of one-dimensional PAGE and two-dimensional PAGE analyses with enzymic deglycosylation and copper-affinity experiments. We found that the identified two main populations of porcine PrP(C) consist of diglycosylated forms and correspond to the full-length (molecular mass 32-36 kDa) and proteolytically modified protein (molecular mass 25-30 kDa), known as C1. The two populations were fully separated during Cu2+-loaded immobilized metal affinity chromatography, indicating different affinity for copper ions. The more basic forms, migrating as species of higher molecular mass, exhibited stronger affinity for copper ions, whereas those with more acidic pI and of lower molecular mass were low-affinity Cu2+-binding molecules and thus could represent N-terminally truncated PrP(C). Size-exclusion chromatography revealed that most of the PrP(C) molecules in porcine brain extracts exist in the form of high-molecular-mass complexes (probably with other proteins). The heterogeneity of porcine PrP(C), resulting from proteolytic modification and glycosylation, influences its ability to assemble into these complexes. N-truncated molecules dominate over full-length PrP(C) in fractions of molecular mass over the range 65-130 kDa, whereas the full-length species are the major forms of PrP(C) present in the monomeric fraction and in complexes above 130 kDa. Two-dimensional PAGE analysis indicated that the complexed PrP(C) differs in the composition of pI forms from the monomers.

Separation of native prion protein (PrP) glycoforms by copper-binding using immobilized metal affinity chromatography (IMAC)

“…autolysis of cells, pH value [15], redox potential etc., may have unpredictable effects on PrP structure and immunogenicity. This is not surprising, since a recent study suggests that the majority of PrP C in pig brain appears in a fully-glycosylated form [38]. We observed that PrP C isolated from uninfected and infected murine brain appeared predominantly to be diglycosylated.…”

Binding of N‐ and C‐terminal anti‐prion protein antibodies generates distinct phenotypes of cellular prion proteins (PrPC) obtained from human, sheep, cattle and mouse

“…Heterogeneity of PrP C proteins is enhanced by endogenous proteolytic modifications, which occurs in vivo [25–27]. PrP C from non‐infected brains consists in addition to full‐length PrP to a significant amount of an N‐terminal truncated PrP C fragment termed C1.…”

“…Copper and heparan sulfate binding have been mediated through its N‐terminal domain [22,23]. However, under physiological conditions the N‐terminal region can be lost by cleavage [23–28]. From endogenous proteolysis, cleavage sites in human PrP C were mapped at amino acids 110–112 and at residues 80–100 generating N‐truncated forms; these are referred to as C1 and C2, respectively.…”

Prion Protein Signaling in the Nervous System—A Review and Perspective

“…In some cells, however, such as neurons in the hippocampus, thalamus, and neocortex, the cytosol form of PrP C is commonly found. 21 Both membrane-bound and soluble forms of PrP C are found in the cerebral spinal fluid. 22 Membrane-bound PrP C can be secreted from cells into the extracellular matrix (ECM) in exosomes, and Martins and coworkers 23 list a number of mechanisms for the release of soluble PrP C from the GPI anchor at the cell membrane.…”

“…215 Further, PrP C may also be involved in other melanocortin diseases such as collagen-related disease (of the eye), 216 pigmented collagen disease of the synovium (involving p53 regulation of monocytes), 217 and thyroid disease. 218 The link between PrP C , αMSH, and NF-κB suggests that the neuroprotective role of PrP C could play a part in the anesthetic response of elderly patients who suffer postanesthetic dementia (Alzheimer's disease), 219,220 possibly involving the role of PrP C in cytoskeleton organization, 21,122 an important focus for further investigation.…”