The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion

Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.     

N-glycosylation sites of plant purple acid phosphatases important for protein expression and secretion in insect cells

Analysis of plant purple acid phosphatases (PAPs) showed high conservation and different distribution of N-glycosylation sites. Oligosaccharide structures of Lupinus luteus acid phosphatase (Lu_AP) produced in insect cells were determined. Mutant Lu_AP and Phaseolus vulgaris (Ph_AP) phosphatases lacking possibility of N-glycosylation at highly conserved sites were generated and expressed in insect cells. A role for N-glycosylation in the stability of PAPs was indicated by unsuccessful attempts to secrete Ph_AP and Lu_AP mutants generated by replacing Asn residues of conserved glycosylation sequons by Ser residues either singly or in combination. We showed that Ph_AP belongs to the group of glycoproteins that require occupancy of all highly conserved glycosylation sites for secretion, whereas replacing of the third position of the glycosylation sequon indicated that Lu_AP may tolerate the absence of some N-glycans. However, the N-glycan located at the polypeptide C-terminus was crucial for secretion of both enzymes. PAP specific activity of glycosylation mutants successfully secreted was similar to the wild-type recombinant proteins.     

Mutational analysis of the N-glycosylation sites of Duffy antigen/receptor for chemokines

The Duffy antigen/receptor for chemokines (DARC) is a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group antigen. The polypeptide chain of DARC contains two NSS motifs at positions 16 and 27 and one NDS motif at position 33 that represent canonical sequences for efficient N-glycosylation. To verify whether all of these three sites are occupied by a sugar chain, we generated mutants in which potential N-glycosylation sites (AsnXSer) were removed by replacement of serine by alanine. Seven DARC glycosylation variants, missing one (S18A, S29A, S35A), two (S18A.S29A, S18A.S35A, S29A.S35A), or three (S18A.S29A.S35A) glycosylation sites, were obtained. cDNA encoding DARC mutants was cloned into the eukaryotic expression vector pcDNA3.1/myc-HisA and expressed in human K562 cells. Stable transfectants expressing wild-type or mutated forms of Duffy were then lysed, purified by metal-affinity chromatography, and subjected to Western blots with an anti-Duffy monoclonal antibody. The gel electrophoresis data indicate that all three canonical sites are used for sugar attachment.     

Using X-ray crystallography of the Asp55Asn mutant of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus to support the mechanistic role of Asp55 as the general base

Because mutations of the ionizable Asp at position 55 of the phosphatidylcholine preferring phospholipase C from Bacillus cereus (PLC(Bc)) to a non-ionizable Asn generate a mutant enzyme (D55N) with 10(4)-fold lower catalytic activity than the wild-type enzyme, we tentatively identified Asp55 as the general base for the enzymatic reaction. To eliminate the alternate possibility that Asp55 is a structurally important amino acid, the X-ray structures of unbound D55N and complexes of D55N with two non-hydrolyzable substrate analogues have been solved and refined to 2.0, 2.0, and 2.3A, respectively. The structures of unbound wild-type PLC(Bc) and a wild-type PLC(Bc)-complex with a non-hydrolyzable substrate analogue do not change significantly as a result of replacing Asp55 with Asn. These observations demonstrate that Asp55 is not critical for the structural integrity of the enzyme and support the hypothesis that Asp55 is the general base in the PLC(Bc)-catalyzed hydrolysis of phospholipids.     

Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity

To investigate the function of N-glycosylation of Cel5A (endoglucanase II) from Hypocrea jecorina, two N-glycosylation site deletion Cel5A mutants (rN124D and rN124H) were expressed in Saccharomyces cerevisiae. The weights of these recombinant mutants were 54 kDa, which were lower than that of rCel5A. This result was expected to be attributed to deglycosylation. The enzyme activity of rN124H was greatly reduced to 60.6% compared with rCel5A, whereas rN124D showed slightly lower activity (10%) than that of rCel5A. rN124D and rN124H showed different thermal stabilities compared with the glycosylated rCel5A, especially at lower pH value. Thermal stabilities were reduced and improved for rN124D and rN124H, respectively. Circular dichroism spectroscopy showed that the modification of secondary structure by mutation may be the reason for the change in enzymatic activity and thermal stability. [BMB Reports 2014; 47(5): 256-261]     

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60 kDa accompanied by a broad band of 60–90 kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53 kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn¹⁶¹ and Asn²⁴¹ on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study.     

Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V

The expressions of integrin alpha5, beta1, and alpha6 were studied in H7721 cells by means of flow cytometric and RT-PCR method after transfected with sense and antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V). The transfected cells were characterized by Northern blot. It was found that the order of expression from high to low was beta1>alpha5>alpha6. Transfection of sense GnT-V up-regulated alpha5 and alpha6, but not beta1 subunit, while antisense GnT-V down-regulated alpha5 and beta1, but not alpha6. The alterations of surface integrin subunits were quite compatible with the changes of their mRNAs. Using enzyme-labeled lectin analysis, it was shown that alpha5 subunit contained only C(2)C(2) biantennary N-glycan, which was not regulated by sense and antisense GnT-V. In contrast, beta1 subunit contained both biantennary and tri-/tetra-antennary N-glycans with GlcNAcbeta1,6Manalpha1,6-branch, and the latter was up- and down-regulated by the sense and antisense GnT-V, respectively. Therefore, the amount of biantennary N-glycans on beta1 subunit, but not the integrin protein, was correlated to the cell adhesion to fibronectin and laminin, which was reduced and elevated in the sense and antisense GnT-V-transfected cells, respectively, as we previously reported.     

Asp and Thr are critical for cation exchange mediated by NhaD, Na/H antiporter of Vibrio cholerae

The Vc-NhaD is an Na⁺/H⁺ antiporter from Vibrio cholerae belonging to a new family of bacterial Na⁺/H⁺ antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu¹⁰⁰Ala, Glu²⁵¹Ala, Glu³⁴²Ala, and Asp³⁹³Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp³⁴⁴Ala, Asp³⁴⁴Asn, and Thr³⁴⁵Ala caused a complete loss of both Na⁺/H⁺ and Li⁺/H⁺ antiport activity whereas the Asp³⁴⁴Glu variant exhibited reduced Na⁺/H⁺ and Li⁺/H⁺ antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na⁺/H⁺ antiport. We discuss the possible role of Asp³⁴⁴ and Thr³⁴⁵ in the functioning of Vc-NhaD.     

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV35S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn189) associated with the point of glycan attachment (Asn189) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35S:prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent Km) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (Ea) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases.     

Partial identification of carbohydrate-binding sites of a Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades by site-directed mutagenesis

The Galalpha1,3Galbeta1,4GlcNAc-specific lectin from the mushroom Marasmius oreades (MOA) contains a ricin B chain-like (QXW)(3) domain at its N-terminus that is composed of three identical subdomains (alpha, beta, and gamma) and a C-terminal domain of unknown function. Here, we investigate the structure-function relationship of MOA to define the number and location of its carbohydrate-binding sites. Based on the sequence alignment of MOA to the ricin B-chain lactose-binding sites, we systematically constructed mutants by site-directed mutagenesis. We have used precipitation and hemagglutination assay for the primary analyses, and surface plasmon resonance for the kinetic analysis. Among amino acid residues at the putative carbohydrate-binding sites, Gln(46) in the alpha subdomain and Trp(138) in the gamma subdomain have been identified to be important amino acid residues directly or indirectly involved in carbohydrate recognition. By surface plasmon resonance, Q46A and W138A were 2.4- and 4.3-fold less active than that of the wild-type MOA (K(a) = 2 x 10(7)), respectively. A double-site mutant (Q46A/W138A) had activity similar to W138A. The C-terminal deletion mutant MOADeltaC showed hemagglutination and precipitation activity, although its binding constant was 12.5-fold less active (K(a) = 1.6 x 10(6)) than that of the wild-type MOA. A C-terminal deletion mutant with mutations at both Gln(46) and Trp(138) (MOADeltaC-Q46A/W138A) was 12,500-fold less active (K(a) = 1.6 x 10(3)) than that of the wild-type MOA. On the basis of this observation, we conclude that both alpha and gamma subdomains are most probably involved in carbohydrate binding, but the beta subdomain appears to be inactive.     

Importance of N-glycosylation positioning for secretion and folding of ovalbumin

To investigate the role of the carbohydrate chain of hen egg ovalbumin (OVA), potential N-glycosylation site-deletion OVA mutants were expressed in yeast. The secretion level of the N292Q and N292/311Q mutants was greatly reduced compared with the wild-type OVA. Furthermore, secretion of the mutants without a carbohydrate chain on Asn-292 could hardly be detected in the culture medium, even if an additional N-glycosylation site was introduced to the OVA molecule. The reduction in secretion level seems to be due to incorrectly folded protein. Moreover, the secretion levels of the wild-type and N311Q mutant reduced in a similar extent as those of the mutants without a carbohydrate chain on Asn-292 in calnexin-disrupted yeast. These results indicate that the carbohydrate chain attached to Asn-292 of OVA has an important role for the secretion and folding in the cells.     

Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A

Peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidases [PNGases (peptide N-glycosidases), N-glycanases, EC 3.5.1.52] are essential tools in the release of N-glycans from glycoproteins. We hereby report the discovery and characterization of a novel bacterial N-glycanase from Terriglobus roseus with an extremely low pH optimum of 2.6, and annotated it therefore as PNGase H⁺. The gene of PNGase H⁺ was cloned and the recombinant protein was successfully expressed in Escherichia coli. The recombinant PNGase H⁺ could liberate high mannose-, hybrid- and complex-type N-glycans including core α1,3-fucosylated oligosaccharides from both glycoproteins and glycopeptides. In addition, PNGase H⁺ exhibited better release efficiency over N-glycans without core α1,3-fucose compared with PNGase A. The facile expression, non-glycosylated nature, unusual pH optimum and broad substrate specificity of this novel type of N-glycanase makes recombinant PNGase H⁺ a versatile tool in N-glycan analysis.     

Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice

Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2–20 μL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine β-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease.     

Effect of N-linked glycosylation on glycopeptide and glycoprotein structure

Asparagine-linked glycosylation is an enzyme-catalyzed, co-translational protein modification reaction that has the capacity to influence either the protein folding process or the stability of the native glycoprotein conjugate. Advances in both glycoconjugate chemical synthesis and glycoprotein expression methods have increased the availability of these once elusive biopolymers. The application of spectroscopic methods to these proteins has begun to illuminate the various ways in which the saccharide affects the structure, function and stability of the proteins.     

Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway

Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the net outcome of the insect cell N-glycosylation pathway.     

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct

Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.     

Unfolded protein response followed by induction of cell death in cultured tobacco cells treated with tunicamycin

When correct folding of protein in the endoplasmic reticulum (ER) is prevented, cells respond to overcome the accumulation of unfolded proteins. This cellular response, which includes the induction of ER chaperones, is called an unfolded protein response (UPR). Although a link between the UPR and apoptosis has been reported in mammalian cells, little is known about this mechanism in plant cells. Asparagine (N)-linked glycosylation of proteins is critical for protein folding in the ER; and tunicamycin, a potent inhibitor of N-linked glycosylation, induces UPR. Growth arrest was observed in cultured tobacco cells treated with tunicamycin. Cell death and induction of Hsr203J, a marker for programmed cell death, were observed in the 24-h period after addition of tunicamycin, following UPR that started within 2 h. These results indicate a strong link between UPR and programmed cell death in plant cells.     

Cysteine 351 is an essential nucleophile in catalysis by Porphyromonas gingivalis peptidylarginine deiminase

Peptidylarginine deiminase (PAD), which catalyzes the deimination of the guanidino group from peptidylarginine residues, belongs to a superfamily of guanidino group modifying enzymes that have been shown to produce an S-alkylthiouronium ion intermediate during catalysis. Thiol-directed reagents iodoacetamide and iodoacetate inactivate recombinant PAD, and substrate protects the enzyme from inactivation. Activity measurements together with peptide mapping by mass spectrometry of PAD modified in the absence and presence of substrate demonstrated that cysteine-351 is modified by iodoacetamide. The pK_a value of the cysteine residue, 7.7 ± 0.2 as determined by iodoacetamide modification, agrees well with a critical pK value identified in pH rate studies. The role of cysteine-351 in catalysis was tested by site-directed mutagenesis in which the cysteine was replaced with serine to eliminate the proposed nucleophilic interaction. Binding studies carried out using fluorescence spectrometry established the structural integrity of the C351S PAD. However, the C351S PAD variant was catalytically inactive, exhibiting <0.01% wild-type activity. These results indicate that Cys 351 is a nucleophile that initiates the enzymatic reaction.     

Chemo-enzymatic synthesis of eel calcitonin glycosylated at two sites with the same and different carbohydrate structures

Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.     

Methods for studying prion protein (PrP) metabolism and the formation of protease-resistant PrP in cell culture and cell-free systems

Transmissible spongiform encephalopathies (TSE) or prion diseases result in aberrant metabolism of prion protein (PrP) and the accumulation of a protease-resistant, insoluble, and possibly infectious form of PrP, PrP-res. Studies of PrP biosynthesis, intracellular trafficking, and degradation has been studied in a variety of tissue culture cells. Pulse-chase metabolic labeling studies in scrapie-infected cells indicated that PrP-res is made posttranslationally from an apparently normal protease sensitive precursor, PrP-sen, after the latter reaches the cell surface. Cell-free reactions have provided evidence that PrP-res itself can induce the conversion of PrP-sen to PrP-res in a highly species- and strain-specific manner. These studies have shed light on the mechanism of PrP-res formation and suggest molecular bases for TSE species barrier effects and agent strain propagation.