Low Mr Form Research Articles

Temperature-dependent phenotypic variation of Campylobacter jejuni lipooligosaccharides

BackgroundCampylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry.ResultsHerein we describe the existence and heterogeneity of multiple LOS forms in C. jejuni strains of human and chicken origin grown at 37°C and 42°C, respectively, as determined on sodium dodecyl sulphate-polyacrylamide electrophoresis gels with carbohydrate-specific silver staining and blotting with anti-ganglioside ligands, and confirmed by nuclear magnetic resonance (NMR) spectroscopy. The C. jejuni NCTC 11168 original isolate (11168-O) was compared to its genome-sequenced variant (11168-GS), and both were found to have a lower-Mr LOS form, which was different in size and structure to the previously characterized higher-Mr form bearing GM1 mimicry. The lower-Mr form production was found to be dependent on the growth temperature as the production of this form increased from ~5%, observed at 37°C to ~35% at 42°C. The structure of the lower-Mr form contained a β-D-Gal-(1→3)-β-D-GalNAc disaccharide moiety which is consistent with the termini of the GM1, asialo-GM1, GD1, GT1 and GQ1 gangliosides, however, it did not display GM1 mimicry as assessed in blotting studies but was shown in NMR to resemble asialo-GM1. The production of multiple LOS forms and lack of GM1 mimicry was not a result of phase variation in the genes tested of NCTC 11168 and was also observed in most of the human and chicken isolates of C. jejuni tested.ConclusionThe presence of differing amounts of LOS forms at 37 and 42°C, and the variety of forms observed in different strains, indicate that LOS form variation may play a role in an adaptive mechanism or a stress response of the bacterium during the colonization of different hosts.

Differential inhibition of high and low Mrthioredoxin reductases of parasites by organotelluriums supports the concept that low Mrthioredoxin reductases are good drug targets

Thioredoxin reductase (TrxR), a NADPH-dependent disulfide oxidoreductase, is vital in numerous cellular processes including defence against reactive oxygen species, cell proliferation and signal transduction. TrxRs occur in 2 forms, a high Mr enzyme characterized by those of mammals, the malaria parasite Plasmodium falciparum and some worms, and a low Mr form is present in bacteria, fungi, plants and some protozoan parasites. Our hypothesis is that the differences between the forms can be exploited in the development of selective inhibitors. In this study, cyclodextrin- and sulfonic acid-derived organotelluriums known to inhibit mammalian TrxR were investigated for their relative efficacy against P. falciparum TrxR (PfTrxR), a high Mr enzyme, and Trichomonas vaginalis TrxR (TvTrxR), a low Mr form of TrxR. The results suggest that selective inhibition of low Mr TrxRs is a feasible goal.

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility

Phosphorylation of rat brain calpastatins by protein kinase C

Calpastatin, the natural inhibitor of calpain, is present in rat brain in multiple forms, having different molecular masses, due to the presence of one (low Mr form) or four (high Mr form) repetitive inhibitory domains. Recombinant and native calpastatin forms are substrates of protein kinase C, which phosphorylates a single serine residue at their N-terminus. Furthermore, both low and high Mr calpastatins are phosphorylated by protein kinase C at the same site. These calpastatin forms are phosphorylated also by protein kinase A, although with a lower efficiency. The incorporation of a phosphate group determines an increase in the concentration of Ca 2+ required to induce the formation of the calpain-calpastatin complex. This effect results in a large decrease of the inhibitory efficiency of calpastatins. We suggest that phosphorylation of calpastatin represents a mechanism capable to balance the actual amount of active calpastatin to the level of calpain to be activated.

Investigation of two glycosylated forms of bile-salt-dependent lipase in human pancreatic juice.

Pure human pancreatic bile-salt-dependent lipase, devoid of its oncofetal glycoform [Mas, E., Abouakil, N., Roudani, S., Miralles, F., Guy-Crotte., O., Figarella, C., Escribano, M. J. & Lombardo, D. (1993) Biochem. J. 289, 609-615], was analyzed on immobilized concanavalin A (ConA). Two variants were separated: an unabsorbed ConA-unreactive fraction; and an absorbed ConA-reactive fraction. Carbohydrate compositions of ConA-reactive and ConA-unreactive fractions were not significantly different, and analysis of 3H-labelled oligosaccharides liberated from these fractions on the ConA-Sepharose column indicated that the fractionation of the bile-salt-dependent lipase on this column depends upon oligosaccharide structures. The activity of the ConA-reactive fraction was however much lower, independent of the substrate (4-nitrophenyl hexanoate or cholesteryl esters), than that of the ConA-unreactive fraction. Therefore, catalytic constants for the hydrolysis of 4-nitrophenyl hexanoate were determined; both fractions had quite similar Km, while the kcat for the ConA-unreactive fraction was 3-4-fold higher than that of the ConA-reactive fraction. ConA-reactive and ConA-unreactive fractions were shown to have slightly different molecular masses and different amino acid compositions. Cleavage patterns after cyanogen bromide treatment of the ConA-reactive and ConA-unreactive fractions suggested that the ConA-reactive (high Mr form) and ConA-unreactive (low Mr form) forms could be different isoforms of the bile-salt-dependent lipase secreted by the human pancreas.

Characterization of NADH-dependent Fe3+-chelate reductases of maize roots

Iron-deficient maize seedlings exhibit a starvation syndrome characterized by an increase in different parameters such as root fresh weight (+ 30%), protein (+ 25%) and plasma membrane-associated NADH Fe3+ −EDTA reductase (NFR; +45%). NFR activity was found associated with 9 000g (20 min) and 110 000 g (1 h) sediments, purified plasma membrane and 110000 g supernatants. No differences were observed between the properties of reductases from Fe-starved versus Fe-sufficient roots. The characterization of NFR was undertaken. Low Mr forms (46 and 28 kDa, as detected by size-exclusion chromatography) were present in all fractions whereas 210 and 110 kDa forms were unique in membranes and 110 000 g supernatants, respectively. The 210 kDa form was solubilized from microsomes and characterized. The enzyme is cetone-resistant and appears to be comprised largely if not totally of the low Mr forms (46 and 28 kDa, corresponding to 30 and 32 kDa bands, respectively, in SDS-PAGE). The 210 kDa form tended to break down to subunits following dilution, and the effect could be prevented by addition of 10% (v/v) glycerol. A three-step purification procedure for microsomal NFR was devised, consisting of acetone fractionation of lysophosphatidycholine solubilized microsomes, Blue Sepharose CL-6B affinity chromatography and a final size exclusion chromatography in the absence of detergent, resulting in a 700-fold purification of the 28 kDa protein. The best electron acceptor for the purified 28 kDa form was ferricyanide (400μmol min−1mg−1 protein) followed by Fe3+−chelates (up to 200μol min−1 mg−1 protein) and other compounds to a lesser extent (cyt c, DCPIP).The 46 kDa form, on the other hand, had high ferricyanide reductase activity (about 300μmol min−1mg−1 protein) and relatively low Fe3+−chelate reductase activity. The properties of NFR (high M, active forms, donor and acceptor specificity, purification behaviour, large hydrophilic domains, size of subunits) suggest a relationship with the NADH-cyt b5 reductase family of FAD-containing proteins. None of the latter flavoproteins is a transmembrane enzyme.

Proteins of the Golgi apparatus

The Golgi marker enzyme, UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase (beta 1-4GalT) was purified 44300-fold in its intact, membrane-bound form from rat liver membranes. The protein was isolated from detergent extracts as a high-M(r) form, having a Stokes radius approximating a globular protein of M(r) 440,000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an M(r) near 51,000, and does not have intermolecular disulfide cross-links. N-terminal sequencing of the enzyme demonstrated that it contains an N-terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5' end of the mRNA [Russo, R. N., Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N-terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low-M(r) forms which were fully catalytically active and which, upon sequencing, were missing a 66-amino-acid stretch from the N-terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with M(r) near 50,000. The N-terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non-protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

Increased levels of a lower molecular weight form of secretory component in cervical scrapes from women with cervical intraepithelial neoplasia.

The protein composition of 'negative' and 'positive' cervical scrapes has been compared using electrophoresis to determine whether differences were present that could be used to pre-select specimens with negative cytology. Only one minor difference in the 75-80 kDa region of the silver-stained patterns was detected in extracts from two well-matched groups. Further studies using 2D-electrophoresis and Western blotting identified the major components in this region as two forms of secretory component (SC) and transferrin. Subsequent blotting of 13 extracts from patients with negative smears and 17 extracts from patients with positive smears indicated very significant differences in the expression of the two forms of SC (P < 0.0002), the 'negatives' having more of the higher Mr form and the 'positives' having more of the lower Mr form. Measuring SC could prove useful for pre-screening cervical scrapes, but more investigations are required to establish the nature of the change and a quantitative method for its detection.

Primary structure of bovine endothelin ETB receptor and identification of signal peptidase and metal proteinase cleavage sites.

A cDNA clone corresponding to the entire coding region of the bovine ETB endothelin receptor mRNA was isolated from a lung cDNA library and sequenced. The cDNA encodes 441 amino acids: 26 constituting an NH2-terminal signal peptide and 415 constituting the mature receptor. The signal peptidase cleavage site was determined by direct amino acid sequencing of purified receptor. A comparison of the predicted amino acid sequence with the available bovine ETA and rat ETB endothelin receptor sequences revealed 63 and 85% homology, respectively. Endothelin receptors of various species are known to be very sensitive to a certain metal proteinase(s) and have been shown to be converted to a lower Mr form in the absence of EDTA. The metal proteinase cleavage site was also determined by direct protein sequencing of the proteolysis product. The amino acid sequence (Ala-Gly-X-Pro-Pro-Arg) surrounding the cleavage site (between Ala-79 and Gly-80) is conserved among the ETB endothelin receptors, explaining the above mentioned proteolytic conversion from the higher to lower Mr forms observed in various species.

Transforming growth factor beta inhibits plasminogen activator (PA) activity and stimulates production of urokinase‐type PA, PA inhibitor‐1 mRNA, and protein in rat osteoblast‐like cells

Transforming growth factor beta (TGF beta) treatment of rat osteoblast-rich calvarial cells or of the clonal osteogenic sarcoma cells, UMR 106-01, resulted in dose-dependent inhibition of plasminogen activator (PA) activity, and increased production of 3.2 kb mRNA and protein for PA inhibitor -1 (PAI-1). Although tissue-type PA (tPA) protein was not measured, TGF beta did not influence production of mRNA for tPA. Production of 2.3 kb mRNA for urokinase-type PA (uPA) was also increased by TGF beta in a dose-dependent manner. The effects of TGF beta on synthesis of mRNA for PAI-1 and uPA were maintained when protein synthesis was inhibited, and were abolished by inhibition of RNA synthesis. Although uPA had not been detected previously as a product of rat osteoblasts, treatment of lysates of osteoblast-like cells with plasmin yielded a band of PA activity on reverse fibrin autography, corresponding to a low Mr form of uPA. Untreated conditioned media from normal osteoblasts or UMR 106-01 cells contained no significant TGF beta activity, but activity could be detected in acidified medium. Treatment of conditioned media with plasmin resulted in activation of approximately 50% of the TGF beta detectable in acidified media. The results identify several effects of TGF beta on the PA-PA inhibitor system in osteoblasts. Net regulation of tPA activity through the stimulatory actions of several calciotropic hormones and the promotion of PAI-1 formation by TGF beta could determine the amount of osteoblast-derived TGF beta activated locally in bone. Stimulation of osteoblast production of mRNA for uPA could reflect effects on the synthesis of sc-uPA, a precursor for the active form of the enzyme.

Intestinal lymph and plasma lipoproteins in the preruminant calf: partial resolution of particle heterogeneity in the 1.040-1.090 g/ml interval.

Our previous studies in the preruminant calf have provided evidence for the heterogeneity of lipoprotein particles in the 1.040-1.090 g/ml density interval in both plasma and postprandial intestinal lymph (Bauchart, D. et al., 1989. J. Lipid Res. 30: 1499-1514; and Laplaud, P. M. et al., 1990. J. Lipid Res. 31: 1781-1792). We therefore attempted to resolve this heterogeneity by use of heparin-Sepharose affinity chromatography. Experiments were performed on three calves; portal vein plasma and intestinal lymph were obtained simultaneously 10 h after a meal, i.e., at peak lipid absorption. In both fluids, the chromatographic profile presented three fractions, I, II, and III. Fraction I was characterized by the presence of cholesteryl ester-rich particles (approximately 35-37% of lipoprotein mass), which migrated electrophoretically as typical high density lipoproteins and exhibited Stokes diameters in the 130-160 A range; apoA-I was the predominant protein. In addition to this polypeptide, fraction II contained small amounts of a supplementary protein (Mr approximately 51,000), exhibiting heparin-binding properties. In the light of results reported in the literature, we suggest that this latter protein could correspond to beta 2 glycoprotein I. The chemical composition of each fraction II closely resembled that of the corresponding fraction I, while their electrophoretic migrations appeared slightly slower and their Stokes diameters slightly larger (155-165 A). Apart from the presence of small amounts of apoA-I, two high Mr proteins (Mr approx. 560,000 and 300,000) were typical of the apolipoprotein moiety of fractions III. The lower Mr form was present as a trace component only in fraction III originating from plasma; its proportion increased in lymph fraction III so as to approximately match that of the higher Mr (i.e., 560,000) protein. In both plasma and lymph, fraction III was electrophoretically heterogeneous, exhibiting a doublet of bands with migration and Stokes diameters (250 A) typical of low density lipoprotein particles. However, no evidence for the presence of a particle resembling lipoprotein[a] in fraction III could be obtained. In lymph only, fraction III contained a supplementary population of lipoproteins with migration intermediary between those of conventional low and high density lipoproteins and with Stokes diameters in the 190-200 A range. Other specific features of lymph fraction III included a sevenfold increase in its triglyceride content (8.5 +/- 3.4% vs. 1.2 +/- 1.1% in the corresponding fraction from plasma), to the detriment of cholesteryl esters, and a higher proportion of protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Ganglioside (GM1)-treated T cells shed CD4

In previous studies (Morrison et al., 1990), we showed that ganglioside (GM1) modulation of CD4 was associated with activation of phospholipase C and increased production of inositol triphosphate, but not with activation of protein kinase C. These results demonstrated a unique signal transduction pathway related to GM1 modulation of CD4 on T cells and raised the question as to whether intracellular Ca2+ levels and related protein kinases would be affected by GM1-induced signalling. We now show that GM1 modulation of CD4 from human T cells corresponds to decreased cellular Ca2+ without significant changes in cellular photein phosphorylation. In the course of this study we discovered that T cells challenged with GM1 exhibited new proteins in their surrounding media. Fractionation of cellular and supernatant proteins show that cells treated with GM1 released proteins with an approximate molecular weight (Mr) of 49 000. This was exclusive of GM1 protein association and GM1-induced protein phosphorylation. Immunoblots demonstrated the presence of CD4 in GM1-treated cell supernatants. Western immunoblots using anti-CD4 antibodies detected a lower Mr form (49 000) of CD4 in the supernatants of GM1-treated cells. These studies further define the unique nature of GM1 signalling relating to modulation of CD4 and demonstrate that the fate of GM1 modulated CD4, in part, involves protein shedding.

Different glycoforms of human thrombomodulin. Their glycosaminoglycan-dependent modulatory effects on thrombin inactivation by heparin cofactor II and antithrombin III.

The relationship between thrombomodulin-associated O-linked glycosammoglycans (GAGs) and the exogenous GAGs heparin or dermatan sulfate was studied in the inhibition of thrombin by antithrombin III (AT III) or heparin cofactor II (HC II). Both rabbit thrombomodulin (TM) and two glycoforms (a high-Mr form containing GAGs and a low-Mr form lacking the majority of O-linked GAGs) of a recombinant human TM deletion mutant (rec-TM) were used. The rapid inactivation of thrombin by HC II in the presence of dermatan sulfate was prevented by both the high-Mr rec-TM and the rabbit TM. In contrast, both rabbit TM treated with chondroitin ABC lyase to remove O-linked GAGs and the low-Mr form of rec-TM had only weak protecting effects. In the absence of exogeneous dermatan sulfate, thrombin inhibition by a high concentration of HC II was slightly accelerated by the high-Mr form of rec-TM but protected by rabbit TM. When thrombin inhibition by AT III in the presence of heparin was studied, both high-Mr rec-TM and rabbit TM again invoked a similar reduction of inactivation rates, whereas in the absence of exogenous heparin, both high-Mr forms accelerated thrombin inhibition by AT III. The diverse reactivities of various forms of TM towards HC II and AT III were also observed during protein C activation by the thrombin-TM complex. These results suggest that thrombin activity at the vessel wall or in fluid phase may undergo major kinetic modulations depending on the type of protease inhibitor, the presence or absence of exogenous GAGs and the glycosylation phenotype of TM. The dependence of TM anticoagulant function on the presence of an intrinsic GAG moiety suggests that variant glycoforms of this endothelial cell cofactor may be expressed differently in a species-, organ-, or tissue-specific manner as a means to regulate TM function in diverse vasculatures.

Purification of recombinant human prostromelysin. Studies on heat activation to give high-Mr and low-Mr active forms, and a comparison of recombinant with natural stromelysin activities

Recombinant human prostromelysin was purified in a single step using Procion Red-Sepharose chromatography. The purified prostromelysin was self-activated to high-Mr (45,000) and low-Mr (28,000) forms by incubation at 55 degrees C without the addition of extraneous activators. The two forms of stromelysin were subsequently separated, again using Procion Red-Sepharose. Both of the heat-activated recombinant forms demonstrated similar specific activities (for the macromolecular substrates casein, gelatin, elastin, proteoglycan and type IV collagen) when compared with either heat- or trypsin-activated natural stromelysin. The heat-activated recombinant stromelysins both showed similar abilities to potentiate activation of human procollagenase when compared with trypsin-activated natural stromelysin.

The octameric structure of β-glucosidase from Botryodiplodia theobromae Pat

1. Whereas only beta-glucosidase A (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced by the tropical fungus Botryodiplodia theobromae Pat. (I.M.I. 115626; A.T.C.C. 26123) in young cultures containing D-cellobiose as carbon source, lower-Mr forms (B, C and D) were found in older cultures when the pH had drifted from the initial value of pH 6.2 to pH 7.9. 2. The Michaelis constants (Km) of the various molecular forms of the enzyme were 0.30 +/- 0.03 mM-, 0.26 +/- 0.01 mM-, 0.20 +/- 0.02 mM- and 0.16 +/- 0.01 mM-o-nitrophenyl beta-D-glucopyranoside for beta-glucosidase forms A (Mr 320,000), B (Mr 160,000), C (Mr 80,000) and D (Mr 40,000) respectively. 3. Only beta-glucosidase D showed substrate inhibition. 4. Only L-arginine was found as the N-terminal residue, and beta-glucosidase A contained 31.7 +/- 0.6 mol of N-terminal L-arginine/mol of the enzyme. 5. Storage of purified beta-glucosidase A under mildly alkaline conditions caused its dissociation into the lower-Mr forms, whereas adjustment of the pH of a solution of beta-glucosidase A to pH 12.0 with 1 M-NaOH led to complete inactivation on incubation at 40 degrees C for 1 h and to the release of 25.2 +/- 1.5 mol of inorganic phosphate/mol of the enzyme. 6. O-Phospho-L-serine was isolated from the acid-hydrolysis product of beta-glucosidase A but not from that of beta-glucosidase D. 7. Reduction and carboxamidomethylation of the various forms of beta-glucosidase gave only one enzymically inactive protein with an Mr of 10,000-11,000. 8. After partial succinylation (3-carboxypropionylation) of beta-glucosidase D at pH 5.0 and removal of the precipitated protein formed, the supernatant solution contained beta-glucosidase components similar to the other molecular forms (A, B and C) and an aggregate (beta-glucosidase Xs) that gave a positive result in the alkaline hydroxylamine test, whereas N-succinylated beta-glucosidase D, an aggregate (form Xp) that behaved like beta-glucosidase Xs and traces of forms A, B and C were found by gel filtration of the solution of the precipitate solubilized at neutral pH (7.0-7.7). 9. These observations are discussed in terms of the proposed octameric structure of beta-glucosidase A based on the result of electron microscopy [Umezurike (1975) Biochem. J. 145, 361-368].

The role of cysteine-150 in the structure and activity of rat liver S-adenosyl-l-methionine synthetase

The present paper reports the tryptic digestion of N-ethylmaleimide-treated S-adenosyl-L-methionine synthetase (high- and low-Mr forms) and the isolation of the modified peptides by h.p.l.c. There is only one site modified after 5 min incubation, and the modification at this site correlates with the main activity decrease. The amino acid composition of this peptide was determined, and its localization in the sequence shows the modified residue as cysteine-150, which is located close to the putative ATP-binding site. Modification of the enzyme for 20 min led to the appearance of a second labelled peptide, which seems to be responsible for about a further 10% of the activity loss. The modification by N-ethylmaleimide of the enzyme was partially prevented in the presence of adenosine 5'-[beta gamma-imido]triphosphate and methionine, further supporting the hypothesis that the modified residues lie within the active site. Urea treatment of the enzyme, followed by modification with N-ethylmaleimide, produces the modification of 7 of the 10 cysteine residues present in the sequence. The results obtained were the same for either of the isoforms.

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface.

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.

Differential glycosylation of the protein (PrP) forming scrapie-associated fibrils

PrP is a glycoprotein found in normal brain. In brain affected by scrapie it forms scrapie-associated fibrils (SAF). PrP from SAF shows considerable heterogeneity of size and charge on two-dimensional gels. It separates into six major regions, the three more acidic regions arising as a result of partial proteolytic degradation. The two more basic higher Mr forms (Mr 34,000 and 29,000) of PrP can be reduced in apparent Mr to a lower Mr form (Mr 25,000) with Peptide-N-glycosidase F. In addition, a series of lectins has been found to bind to PrP. Some bind preferentially to the higher Mr forms whereas others bind more strongly to the lower Mr form. Some of the heterogeneity of PrP is therefore due to differential N-glycosylation. We suggest that one or two N-linked carbohydrate chains are bound to the protein causing some of the differences in Mr. The major cause of heterogeneity of PrP is therefore proteolytic cleavage combined with differential glycosylation at the two potential N-glycosylation sites. The glycolipid moiety attached to PrP may be responsible for some lectin binding to all three bands. Using lectins as a probe to study potential differences in N-glycosylation we have looked at their binding to PrP isolated from SAF, from different strains of scrapie and from different regions of the same brain. No major differences in the N-glycan moieties were found.

Identification of Different Forms of Human C4b-Binding Protein Lacking β-Chain and Protein S Binding Ability

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.

Receptor-Mediated Endocytosis and Differential Synthesis of Mannose 6-Phosphate Receptors in Isolated Spermatogenic and Sertoli Cells*

Mannose 6-phosphate (Man 6-P) receptors participate in the targeting of both newly synthesized and extracellular acid hydrolases to lysosomes, and also may mediate the effects of a number of growth factors. In this study, Man 6-P receptors were isolated from [35S]methionine-labeled germ cells and Sertoli cells by phosphomannan-Sepharose affinity chromatography and were analyzed by polyacrylamide gel electrophoresis and autoradiography. Mouse pachytene spermatocytes and round spermatids isolated by unit gravity sedimentation synthesized predominantly the 46,000 mol wt (Mr) cation-dependent Man 6-P receptor and only low levels of the 270,000 Mr cation-independent Man 6-P receptor. In contrast, Sertoli cells synthesized substantial amounts of the cation-independent Man 6-P receptor, but little of the cation-dependent receptor. To determine if these receptors function on the cell surface, we have monitored Man 6-P receptor-mediated endocytosis in isolated germ cells and Sertoli cells. In pachytene spermatocytes and round spermatids, [125I]Man 6-P-bearing ligands were internalized and processed to lower Mr forms by an endocytic mechanism that was time dependent, proportional to cell number, and inhibited by Man 6-P. Like germ cells, Sertoli cells in primary culture endocytosed radiolabeled Man 6-P-bearing ligands at levels that were about 10% of the endocytic activity measured for 3T3 fibroblasts. This low endocytic activity correlates with the levels of the cation-independent Man 6-P receptor synthesized by germ cells, but not with the higher levels synthesized by Sertoli cells. Membrane binding assays verified the high steady state levels of the cation-independent Man 6-P receptor in Sertoli cells, suggesting that the low endocytic activity detected in these cells may result from restricted expression of the cation-independent receptor on the cell surface. These results indicate that both spermatogenic and Sertoli cells have surface Man P-6 receptors capable of mediating endocytosis. However, these cells exhibit marked differences in the expression of the cation-independent and -dependent Man 6-P receptors, perhaps reflecting differential roles of these receptors in protein trafficking and/or intercellular communication during germ cell differentiation.