Phosphomannose binding proteins: the phosphomannosyl receptor and insulin like growth factor II receptor

Proteins that bind phosphomannose residues in glycoproteins exhibit widely different functions. They are found as receptors of lysosomal enzymes, as ligatin that binds peripheral glycoproteins and as a lectin in parasites. The identity of the phosphomannosyl receptor for lysosomal enzymes and the insulin like growth factor II receptor raises interesting questions regarding their function.     

Radiometric assays of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety

The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety.     

Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts

The 215-kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand-receptor interaction. Rather the antibody appears to cross-link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of "trapped receptors" is enhanced (t1/2 = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t1/2 = 30 min).     

Human brain creatine kinase binding to immobilized cibacron blue F3GA: characterization and use in purification of the enzyme.

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) from adult human brain grey matter was purified by cibacron blue F3GA-Sepharose affinity chromatography. By gel electrophoresis of the purified enzyme under non-denaturing conditions a single protein band was observed. The dye-bound enzyme was eluted using its substrate, ATP. Reversibility of the binding of purified creatine kinase to blue Sepharose by ATP in a concentration-dependent manner indicated that the cibacron blue molecule which structurally mimics nucleotides occupied the substrate binding site of the enzyme. Also the marked dependence of enzyme binding to blue Sepharose on Mg2+ concentration suggested that Mg2+ ion is capable of combining with the dye moiety to form a site-specific binding complex that is similar to the physiological substrate of creatine kinase, namely Mg(2+)-ATP or Mg(2+)-ADP.     

Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.     

Butanedione treatment reduces receptor binding of a lysosomal enzyme to cells and membranes

Treatment of the lysosomal enzyme, α-L-iduronidase, with 2,3 butanedione, an arginine modifying reagent, under conditions where enzyme activity was unaffected, reduced by 50% the internalization of the enzyme into cultured human fibroblasts. The lowered rate of internalization was a result of a reduced binding of the enzyme to cell surface receptors. The butanedione treatment of α-L-iduronidase caused a 90% reduction of binding when isolated fibroblast membranes were used as the source of receptor. This marked reduction of binding was also seen when membranes from a rat chondrosarcoma were examined. Although there is ample evidence that the receptor recognizes mannose 6-phosphate residues on the enzyme, the results suggest that other structural features, such as arginine moieties, may also be important in iduronidase binding.     

Processing of the phosphorylated recognition marker in lysosomal enzymes. Characterization and partial purification of a microsomal alpha-N-acetylglucosaminyl phosphodiesterase

N-Acetylglucosamine(1)phospho(6)mannose groups recently identified in lysosomal enzymes were proposed to be precursors of the recognition markers terminating with mannose 6-phosphate (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 225, 6633-6639; Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura, K. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7074-7078). To study the presumptive enzyme removing N-acetylglucosamine from the diester, an assay was developed using a radioactive oligosaccharide containing diester groups of the above structure. An alpha-N-acetylglucosaminyl phosphodiesterase cleaving this substrate in vitro was found in human placenta and in rat liver. The enzyme was solubilized from the microsomal fraction of human placenta and more than 800-fold purified with 75% yield. It is distinct from the lysosomal alpha-N-acetylglucosaminidase by the criteria of immunological cross-reactivity, substrate specificity, and heat stability. The partially purified enzyme cleaves alpha-N-acetylglucosamine phosphodiester bonds in oligosaccharides from lysosomal enzymes, in lysosomal enzymes, and in UDP-N-acetylglucosamine. We propose that the microsomal alpha-N-acetylglucosaminyl phosphodiesterase is involved in the processing of the phosphorylated recognition marker in lysosomal enzymes.     

Human cytomegalovirus binding to fibroblasts is receptor mediated.

The binding of radiolabeled human cytomegalovirus (HCMV) strain AD169 to human lymphocytes, lymphoblastoid cell lines, monocytes, and fibroblasts varied over a 20-fold range. Since maximum binding was observed with human foreskin fibroblasts (HFF), interactions of radiolabeled HCMV with this cell type were analyzed quantitatively. Binding of HCMV to HFF at 4 degrees C was specific and saturable; at low viral inputs specific binding averaged 16.4% of input and nonspecific binding was less than 1% of input. Binding curves yielded single-component linear Scatchard plots indicating an average Kd of 1.1 nM and 5,262 available virus-binding sites per cell. A two-component Scatchard curve was obtained at 37 degrees C and reflected viral internalization, since it could be converted to a single-component curve by the use of paraformaldehyde-fixed cells. HCMV strain Towne was found to bind to the receptor used by HCMV strain AD169 with similar affinity. HCMV failed to bind to protease-treated HFF or to HFF grown in the presence of inhibitors of glycosylation. Sialic acid residues, however, were not found to be important in binding. These data indicate that a single type of molecule, likely a glycoprotein, on the surface of HFF serves as a specific receptor for the virus.     

Effects of fluid shear on immobilized enzyme kinetics.

There have been a number of reports concerning the damaging effects of shear on globular proteins in solution. Some recent work has indicated, however, that globular proteins in solution are relatively stable, but may be inactivated at air-liquid interfaces during shearing. This study investigated the effects of fluid shear on immobilized enzyme activity. Immobilized enzyme reactors were built to operate with the enzyme immobilized at the boundary of a fluid flow field. Two different enzymes, penicillinase and lactate dehydrogenase, were covalently bound to the interior surface of nylon tubes. Fluid shear rate was changed by varying the flow rate of substrate (reactant) solution through the tube, and fluid shear stresses were increased by increasing the viscosity of the recirculating solution. There were no observed effects of fluid shear on immobilized penicillinase or lactate dehydrogenase activity at shear rates of up to 10,350 s-1 or at shear stresses of up to 73 Pa.     

Mechanism for ganglioside-mediated modulation of a calmodulin-dependent enzyme. Modulation of calmodulin-dependent cyclic nucleotide phosphodiesterase activity through binding of gangliosides to calmodulin and the enzyme.

Gangliosides were recently shown to bind to calmodulin (Higashi, H., Omori, A., and Yamagata, T. (1992) J. Biol. Chem. 267, 9831-9838). This prompted us to investigate the effects of gangliosides on the calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. Several species of gangliosides competitively inhibited calmodulin-stimulated phosphodiesterase activity, with GD1b, GT1b, and GD1a being noted to do so particularly (group 1). GM1, GQ1b, and GM2 (group 2) were less inhibitory, and GM3, GM3(NeuGc), GalCer, sulfatide, GgOse4Cer, and oligosaccharide portions of inhibitory gangliosides showed no inhibition in accordance with the binding specificity of calmodulin to gangliosides. Trypsin-activated phosphodiesterase was inhibited by gangliosides with similar specificity, indicating interactions of gangliosides with the enzyme. Inhibition, however, was less than that of calmodulin-dependent activity by these compounds and, in both cases, was eliminated by excess calmodulin. In the absence of calmodulin, group 1 gangliosides at lower concentrations activated the intact enzyme but inhibited it over a certain range of increase in concentration. Ganglioside-dependent modulation of calmodulin-dependent phosphodiesterase activity is thus shown to be due to interactions of gangliosides with both calmodulin and the enzyme, and consequently, ganglioside-calmodulin binding is likely the mechanism for regulation of the enzyme.     

Molecular size of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase as determined in situ in Golgi membranes by radiation inactivation.

The radiation inactivation method was used to determine the molecular size of the two enzymes that participate in the synthesis of the phosphomannosyl recognition marker of lysosomal proteins. The determinations were carried out in situ, in Golgi membranes isolated from normal human placenta and cultured skin fibroblasts. A molecular size of 228 +/- 29 kDa was found for placental N-acetylglucosaminyl-phosphotransferase, and 129 +/- 11 kDa for placental alpha-N-acetylglucosaminyl phosphodiesterase. The values for the fibroblast enzymes were about 20% higher, 283 +/- 27 kDa and 156 +/- 14 kDa for the transferase and phosphodiesterase respectively. Triton X-100 had no effect on the molecular size of these enzymes.     

Explanation of anomalous binding kinetics with a high yield immobilized enzyme system

The activities of glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) and catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) from commercial preparations do not give typical adsorption curves upon immobilization on non-porous polyethylenimine-coated glass microbeads. The cause of this effect with glucose oxidase was investigated. Protein binding exhibited a rectangular hyperbolic adsorption isotherm, approaching saturation at high concentrations, however, enzyme activities did not. The isotherm for activities exhibited a maxima which corresponded to less than 50% saturation with regard to total protein adsorption. The enzyme preparation was found to contain small quantities of several low molecular weight impurities as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These impurities apparently compete with glucose oxidase for binding. When large excesses of protein are added to beads, the binding of impurities becomes significant and the amount of enzyme activity per unit of bead is reduced.     

Studies of the binding of ethidium bromide to cells before and after enzyme treatment.

Binding of ethidium bromide (EB) to cells before and after HCl, pepsin and RNase treatment was investiaged by spectophotometric and fluorimetric methods. Binding isotherms, calculated with the McGheevon Hippel equation, taking EB as a non-interacting ligand, revealed the influcence of these treatments on the fluorescence characteristics of the cells which were measured by flow-through cytofluorimetry. Thus pepsin- and RNase-treated cells have a reduced intercalation capacity due to the loss of cytoplasmic RNA and RNA hydrolysis, respectively. HCl alone, or in association with pepsin, increased the equilibrium constant K considerably. Thus at low free EB concentrations the enchanced EB affinity of acid-pretreated cells generates a high fluorescence intensity, by comparison with treatments at neutral pH. This result contradicts the interpretation of high EB binding to acid pretreated cells which is commonly believed to be due to hydrolytic histone removal from potential intercalation sites. With increasing free EB concentrations the fluorescence intensities of RNase- and pepsin-treated cells culminate at the same level due to their amost identical intercalation capacities. Consequently, quantitative DNA analysis of pretreated cell suspensions with EB can only be performed if the alteration, induced by the pretreatment, has previously been studied.     

Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase.

The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.     

Effects of common radioiodination procedures on the binding of glycoproteins to immobilized lectins

Representative glycoproteins including fetuin, protein A, ovalbumin, α₁ acid glycoprotein, and the major glycoprotein of equine infectious anemia virus were labelled with ¹²⁵I by the chloramine-T or Bolton-Hunter procedure and their binding to immobilized Con A or lentil lectin compared to untreated samples of each glycoprotein. Glycoprotein modification was no greater than one substituted residue per protein molecule. Yet the radioiodinated glycoproteins typically displayed only 0–50% of the lectin binding observed with untreated samples. These results indicate that lectin glycoprotein binding can be markedly altered by minor modifications in protein structure.     

Lectin binding to rat spermatogenic cells: Effects of different fixation methods and proteolytic enzyme treatment

Summary The binding of a panel of eight different fluorescein-conjugated lectins to rat spermatogenic cells was investigated. Particular attention was paid to the effects of different fixation methods and proteolytic enzyme digestion on the staining pattern.Concanavalin A (Con A), wheatgerm agglutinin (WGA), succinylated WGA (s-WGA) and agglutinin from gorse (UEA I) stained the cytoplasm of most germ cells as well as the spermatid acrosome. In contrast, peanut agglutinin (PNA), castor bean agglutinin (RCAI) and soy bean agglutinin (SBA) mainly stained the acrosome. The staining pattern varied depending on the fixation method used. PNA was particularly sensitive to formalin fixation, while SBA, DBA and UEA I showed decreased binding and Con A, WGA, s-WGA and RCA I were insensitive to this type of fixation. Pepsin treatment of the sections before lectin staining caused marked changes in the staining pattern; staining with PNA in formalin-fixed tissue sections was particularly improved but there was also enhanced staining with SBA and horse gram agglutinin (DBA). On the other hand, in Bouin- and particularly in acetone-fixed tissue sections, pepsin treatment decreased the staining with several of the lectins, for example WGA and UEA I.     

Coupling of glucagon receptor to adenylyl cyclase. Requirement of a receptor-related guanyl nucleotide binding site for coupling of receptor to the enzyme.

Effects of glucagon and guanyl nucleotides on the rat liver plasma membrane adenylyl cyclase were studied. It was established that: 1) glucagon stimulates the fully guanyl-5'-yl imidodiphosphate (GMP-P(NH)P)-activated enzyme between 20 and 70%, provided a guanyl nucleotide is present in the assay; 2) glucagon has no effect on adenylyl cyclase activity in membranes activated fully by GMP-P(NH)P and then washed free of nucleotides. It is concluded that occupancy of the guanyl nucleotide binding site that activates the catalytic moiety of the system is not sufficient to promote hormone-receptor coupling to adenylyl cyclase and that occupancy of a second site by guanyl nucleotides is essential to effect stimulation of adenylyl cyclase by the glucagon-receptor complex. The data presented raise the question whether the guanyl nucleotide site that promotes coupling is distinct from the guanyl nucleotide site that modulates binding of glucagon to receptor and whether the occupancy of the guanyl nucleotide site associated with the catalytic moiety is necessary for coupling.     

Lysosomal enzyme binding receptor protein from monkey brain and its phosphorylation.

The lysosomal enzyme binding receptor protein isolated from monkey brain by phosphomannan-Sepharose affinity chromatography was phosphorylated by [gamma-32P] ATP by protein kinases tightly associated with the receptor protein. A greater than 200 kDa protein was phosphorylated on both serine and tyrosine residues and a approximately 45 kDa protein was phosphorylated on only serine residues as evidenced by SDS-gel electrophoresis, autoradiography and phosphoamino acid analysis [(Panneerselvam, Ramamoorthy & Balasubramanian (1987) Biochem Biophys Res Commun, 147, 927-935)]. 125I-labelled lysosomal enzymes could be cross-linked to the receptor protein in the presence of disuccinimidyl suberate. Phosphorylation of the receptor on both serine and tyrosine residues was inhibited by quercetin, polylysine and polymyxin B. Catalytic subunit of cyclic AMP-dependent protein kinase preferentially phosphorylated the approximately 45 kDa protein. In the presence of Triton X-100, phosphorylation of a few additional protein bands on non-tyrosine residues was observed. There was a marked reduction in the efficiency of binding lysosomal enzymes by the phosphorylated receptor protein in comparison to the unphosphorylated receptor protein.