Wt Precursor Research Articles

Oligodendrocytes Enforce Immune Tolerance of the Uninfected Brain by Purging the Peripheral Repertoire of Autoreactive CD8+ T Cells

Myelin-specific CD8(+) Tcells are thought to contribute to the pathogenesis of multiple sclerosis. Here we modeled this contribution in mice with CD8(+) Tcells recognizing ovalbumin (OVA) expressed in oligodendrocytes (ODCs). Surprisingly, even very high numbers of activated OVA-reactive CD8(+) Tcells failed to induce disease and were cleared from the immune system after antigen encounter inthe centralnervous system (CNS). Peripheral infection with OVA-expressing Listeria (Lm-OVA) enabled CD8(+) Tcells to enter the CNS, leading to the deletion of OVA-specific clones after OVA recognition on ODCs. In contrast, intracerebral infection allowed OVA-reactive CD8(+) Tcells to cause demyelinating disease. Thus, in response to infection, CD8(+) Tcells also patrol the CNS. If the CNS itself is infected, they destroy ODCs upon cognate antigen recognition in pursuit of pathogen eradication. In the sterile brain, however, antigen recognition on ODCs results in their deletion, thereby maintaining tolerance.

The antitumor monoclonal antibody 806 recognizes a high‐mannose form of the EGF receptor that reaches the cell surface when cells over‐express the receptor

Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2-7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain. mAb 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2-7 EGFR and a subset of the wild-type (wt) EGFR when overexpressed, but does not bind the EGFR expressed in normal tissues. Recently, we demonstrated that the mAb 806 epitope is restricted to a short cysteine loop of the EGFR (amino acids 287-302) that is only available for antibody binding in a transitional form of the receptor, which occurs as the receptor changes from its inactive tethered conformation to a dimeric untethered form. The truncation associated with the de2-7 EGFR mutation renders this receptor constitutively untethered, leading to increased binding of mAb 806. We now show that mAb 806 preferentially binds the immature high-mannose wt and de2-7 EGFR precursors normally located in the endoplasmic reticulum, indicating that this form of the wt EGFR is also constitutively untethered. Using the unique specificity of mAb 806, we clearly demonstrated the presence of these high-mannose EGFR precursors on the cell surface. Given that the high-mannose forms of the wt EGFR must be untethered they may contribute to the spontaneous EGFR signaling reported in cells overexpressing the receptor. These precursor forms of the EGFR thus represent novel tumor targets and contribute to the exceptional selectivity of mAb 806 for EGFR when overexpressed in cancer cells. As our observations are likely to apply to other receptors overexpressed in cancer, they suggest a strategy for developing antitumor antibodies even when the target receptor is expressed in normal tissue.

A murine model of autosomal dominant neurohypophyseal diabetes insipidus reveals progressive loss of vasopressin-producing neurons.

Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disorder caused by mutations in the arginine vasopressin (AVP) precursor. The pathogenesis of FNDI is proposed to involve mutant protein-induced loss of AVP-producing neurons. We established murine knock-in models of two different naturally occurring human mutations that cause FNDI. A mutation in the AVP signal sequence [A(-1)T] is associated with a relatively mild phenotype or delayed presentation in humans. This mutation caused no apparent phenotype in mice. In contrast, heterozygous mice expressing a mutation that truncates the AVP precursor (C67X) exhibited polyuria and polydipsia by 2 months of age and these features of DI progressively worsened with age. Studies of the paraventricular and supraoptic nuclei revealed induction of the chaperone protein BiP and progressive loss of AVP-producing neurons relative to oxytocin-producing neurons. In addition, Avp gene products were not detected in the neuronal projections, suggesting retention of WT and mutant AVP precursors within the cell bodies. In summary, this murine model of FNDI recapitulates many features of the human disorder and demonstrates that expression of the mutant AVP precursor leads to progressive neuronal cell loss.

Muscarinic cholinergic and glutamatergic reciprocal regulation of expression of hippocampal cholinergic neurostimulating peptide precursor protein gene in rat hippocampus

Hippocampal cholinergic neurostimulating peptide, an undecapeptide originally isolated from the hippocampus of young rats, enhances acetylcholine synthesis in rat medial septal nucleus in vitro. Hippocampal cholinergic neurostimulating peptide is derived from the N-terminal region of its 21-kmol.wt precursor protein. The highest expression of the hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA is in hippocampal pyramidal neurons. In an in vitro rat hippocampal slice, preparation in which electrical stimulation could be delivered to the Schaffer collateral-CA1 pyramidal cell synapse, semi-quantitative non-radioisotopic in situ hybridization, demonstrated that expression of the hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA is regulated by neuronal activity. Selective inhibition with pharmacological agents revealed that the constitutive hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA level can be up-regulated by d-(−)-2-amino-5-phosphono-valeric acid, and that activity-dependent transcription can be inhibited by tetrodotoxin, nifedipine, 6-cyano-7-nitroquinoxaline-2,3-dione, and scopolamine, but not by mecamylamine. These results indicate that septal cholinergic neurons and hippocampal glutamatergic neurons exert a reciprocal influence over the expression of hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA in the hippocampus, and that the activity-dependent and constitutive expressions of hippocampal cholinergic neurostimulating peptide precursor protein messenger RNA may be regulated by different routes, involving calcium influx via l-type Ca 2+ channels and N-methyl- d-aspartate receptors.

Lipoprotein receptor-related protein in brain and in cultured neurons of mice deficient in receptor-associated protein and transgenic for apolipoprotein E4 or amyloid precursor protein

The role of the receptor-associated protein in controlling the expression of the low-density lipoprotein receptor-related protein was analysed in brain and in cultured neurons of receptor-associated protein −/− mice. In addition, the effect of two important ligands of lipoprotein receptor-related protein in brain, i.e. apolipoprotein E and amyloid precursor protein, was examined by crossing the receptor-associated protein −/− mice with transgenic mice overexpressing these proteins specifically in neurons. The immunohistochemical localization of lipoprotein receptor-related protein and receptor-associated protein in wild-type mouse brain was demonstrated to be congruent over all structures, including the cortex and hippocampus. In primary hippocampal neurons, lipoprotein receptor-related protein was distributed somatodendritically and receptor-associated protein was concentrated perinuclearly. In hippocampal neurons from receptor-associated protein −/− mice, lipoprotein receptor-related protein was redistributed over the cell body at the expense of the dendrites. In the absence of receptor-associated protein, maturation of lipoprotein receptor-related protein is slow, resulting in accumulation of the uncleaved 600,000 mol. wt precursor. Neither the added expression of apolipoprotein E4 nor that of amyloid precursor protein in cultured neurons influenced the maturation of lipoprotein receptor-related protein, in either the presence or absence of receptor-associated protein. This result shows that receptor-associated protein is not needed to allow co-expression of lipoprotein receptor-related protein with these ligands in neurons. Furthermore, the typical ramified neuronal morphology of cultured primary neurons and the histology and architecture of the brain were normal in receptor-associated protein −/− mice and in all of the double transgenic mice. Finally, we demonstrated that the survival of receptor-associated protein −/− hippocampal neurons was normal and unaffected by the genotype of the glial feeder cells, whether they were derived from wild-type mice or from mice deficient in receptor-associated protein or apolipoprotein E. These results show that, despite the dramatic effect on maturation and cellular localization of lipoprotein receptor-related protein, the absence of receptor-associated protein did not result in any notable physiological, functional or morphological effects.

Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins.

This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) (wild type)] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product.

In vitro evidence for atrial natriuretic factor-(5-28) production by macrophages of adult rat thymi.

Although recent evidence suggests the presence of the 15-kilodalton (K) mol wt (Mr) precursor of atrial natriuretic factor (ANF) and pro-ANF mRNA in the thymus of human and rat neonates, the cellular origin of the peptides in the tissues remains to be elucidated. We report here that in adult male rats, the 15K M(r) presumptive precursor for ANF and a smaller 3K M(r), N-terminal truncated congener of the peptide, ANF-(5-28), are localized in a small population of thymic macrophages. The production of ANF in the thymus was further confirmed by demonstrating the presence of a single band of pro-ANF mRNA signal of approximately 0.8 kilobases from the tissue extract, corresponding to that in the heart. To examine the distribution of pro-ANF mRNA at a cellular level, colorimetric in situ hybridization with digoxigenin-labeled 30-mer oligonucleotides complementary to the first 10-amino acid sequence of ANF-(1-28) was employed. Positive staining was found in thymic cells localized mainly in subcapsular areas, with diffuse staining of positive cells in both the cortex and medulla mainly concentrated around the cortico-medullary junction of the tissue. The identity of the ANF-positive cells was further investigated using a double staining technique to costain immunoreactive (ir) ANF and the macrophage markers ED1 and S22 or the T-cell marker OX-19 in both thymic sections and in monolayer cultures of thymic cells. In the latter, approximately 17% of the adherent cells stained positive for irANF after 48 h in culture. Of these cells, more than 95% also stained positive for the macrophage marker ED1, whereas approximately 36% of the ED1-positive cells were colocalized with irANF. In contrast, no irANF-positive cells were colocalised with the pan-T-cell marker OX-19. In tissue sections, irANF was found in cells distributed predominantly around the cortico-medullary junctions and subcapsular areas, with diffuse staining of the cells in the medullary and cortical regions. Sephadex G-50 gel chromatographic profiles of thymic extracts revealed a major peak of immunoreactivity consistent with 15K M(r) and a smaller peak that coeluted with rat ANF-(1-28) of 3K M(r). HPLC analysis of the 3K M(r) species showed a single peak of immunoreactivity, which eluted with a retention time identical to that of synthetic rat ANF-(5-28).(ABSTRACT TRUNCATED AT 400 WORDS)

Investigation of the structural requirements for peptide precursor processing in AtT-20 cells using site-directed mutagenesis of proadrenocorticotropin/endorphin.

Mouse pro-ACTH/endorphin (or POMC) contains in its sequence each of the four possible pairs of basic amino acids recognized as potential cleavage sites in the production of bioactive peptides from higher mol wt precursors: KR (lysine-arginine), RR, RK, and KK. To examine the structural requirements for processing and routing in one region of pro-ACTH/endorphin, a reporter mutation was introduced into the mouse pro-ACTH/endorphin cDNA; a methionine residue was mutated to an isoleucine residue to allow biosynthetic double labeling with [3H]Ile and [35S]Met. Analysis of stable cell lines expressing the reporter cDNA indicated that this mutation did not affect processing or secretion. Therefore, additional mutations were introduced on the reporter background to investigate important structural features of the precursor. First, the tripeptide signal for N-linked glycosylation in the N-terminal glycopeptide (Asn65,Ser66,Ser67) was disrupted by the conservative substitution of asparagine65 with a glutamine residue. Secondly, O-glycosylation was prevented by substitution of threonine45 with an alanine residue. Finally, lysine50 was mutated to an arginine residue, transforming the RK doublet preceding the gamma 3MSH sequence into an RR doublet. The results show that the enzymatic machinery of AtT-20 cells fails to cleave efficiently at the Arg-Lys (RK) site even after elimination of any possible structural hindrance by carbohydrate side-chains. Elimination of O-linked oligosaccharides to the N-terminal side of gamma 3MSH did not allow cleavage at the RK site, and elimination of N-linked oligosaccharides did not alter the processing and routing of pro-ACTH/endorphin in AtT-20 cells. However, mutation of the RK sequence to RR allowed extensive cleavage regardless of the occurrence of O- or N-glycosylation.

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.

Biogenesis of the yeast lysosome (vacuole): biosynthesis and maturation of proteinase yscB.

The biosynthesis and processing of the vacuolar (lysosomal) proteinase yscB was followed in vivo and in vitro. In vitro transcription--translation of the cloned proteinase yscB gene results in a high mol. wt precursor protein (HMr-precursor) of Mr = 73,000. In vivo a precursor of identical mol. wt is found in sec6l mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum (ER). In contrast to N-glycosylated intermediate mol. wt forms of proteinase yscB, the HMr-precursor of the enzyme is not N-glycosylated, indicating its appearance outside the ER. sec18 Mutant cells, wild type for translocation of proteins into the ER but blocked in the delivery step of secretory proteins to the Golgi apparatus, accumulate a lower mol. wt precursor (LMr-precursor) of proteinase yscB of Mr = 41,500. Processing of the HMr-precursor of proteinase yscB to the LMr-precursor requires proteolytic cleavage which is independent of the proteinase yscA gene product, a protein known to be involved in the processing event of the LMr-precursor of proteinase yscB to the mature enzyme of Mr = 33,000. A LMr-precursor of proteinase yscB of Mr = 42,000 accumulates in proteinase yscA-deficient mutant cells (allele pep4-3). This form is enzymatically active. Incubation in vitro of the LMr-precursor of proteinase yscB with mature proteinase yscA leads to further activation and to processing of the enzyme yielding the mature 33,000 Mr protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y and its flanking peptide in human endocrine tumors and plasma.

Neuropeptide Y (NPY) and its flanking peptide (CPON) were measured in extracts of pheochromocytomas (n = 26), ganglioneuroblastomas (n = 8), and other tumors (n = 95) and plasma (n = 38). NPY was present in high concentrations in the majority of the pheochromocytomas, but was absent in 3 tumors. Similar concentrations of NPY were measured using N- and C-terminal-directed antisera, and there was a good correlation between the concentrations of NPY immunoreactivity and CPON immunoreactivity in the same extracts. Gel permeation chromatography revealed that the separate peptides NPY and CPON were the major products. No significant amounts of a large mol wt precursor containing both immunoreactivities was found. Among the other tumors, 6 of 22 carcinoid tumors, 2 of 2 somatostatinomas, and 3 of 18 insulinomas contained detectable amounts of NPY. Plasma NPY concentrations were very low in normal subjects, and extraction and a 10-fold concentration step were necessary to establish the normal range (0.35-1.25 pmol/L). Plasma NPY concentrations were detectable in unextracted plasma (10 pmol/L) in 50% of patients with pheochromocytomas, but no correlation was found between plasma NPY concentrations and either plasma norepinephrine or epinephrine concentrations. These results indicate that 1) NPY and CPON are present in most, but not all, pheochromocytomas and ganglioneuroblastomas; 2) secretion of NPY immunoreactivity may be independent of that of catecholamines; 3) CPON immunoreactivity is present in plasma of patients with pheochromocytoma; 4) the majority of NPY and CPON immunoreactivities appears to be in the form of the separate free peptide, and there is very little precursor containing both immunoreactivities expressed in these tumors; and 5) NPY immunoreactivity is present in a variety of other endocrine tumors.

Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL-60: application to the study of myeloid lysosomal enzyme synthesis and processing

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.

Separation and Analysis of Subcellular Organelles in a Human Promyelocytic Leukemia Cell Line, HL-60: Application to the Study of Myeloid Lysosomal Enzyme Synthesis and Processing

We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins.

Biosynthesis of a Novel Thyroxine-Binding Protein (27K Protein) in Human Hepatoma (Hep G2) Cells*

Human hepatoma (Hep G2) cells were shown to synthesize and secrete a novel T4-binding protein, called 27K protein for its apparent mol wt on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The mRNA coding for this protein was characterized by immunoprecipitation of [125I]T4 bound to 27K protein secreted into the medium of oocytes injected with total Hep G2 RNA. Sucrose gradient fractionation of RNA from Hep G2 cells showed that TBG mRNA and 27K mRNA had different sizes, indicating that TBG and 27K protein are two distinct proteins. In vitro translation of RNA in a rabbit reticulocyte lysate demonstrated that the translation product immunoprecipitated by anti-27K serum had the same mol wt as the immunoprecipitated protein from whole cells labeled with [35S]methionine, thus suggesting that 27K protein is neither derived from TBG nor synthesized through a larger mol wt precursor, and also that it does not contain carbohydrates. The absence of carbohydrates was further supported by the observation that [3H]mannose was not covalently bound to the 27K protein when Hep G2 cells were labeled with [3H]mannose, nor was there a shift in apparent mol wt when the cells were treated with the glycosylation inhibitor tunicamycin. The kinetics of secretion of 27K protein were similar to those of albumin and faster than those of TBG, which is also in keeping with the nonglycoprotein nature of 27K protein.

Constitutive Biosynthesis of Bone Gla Protein in a Human Osteosarcoma Cell Line*

A human osteosarcoma cell line derived from cells obtained from a patient with Paget's disease is shown to synthesize and secrete bone Gla protein (BGP); (osteocalcin), a noncollagenous bone matrix protein. Using a human BGP-specific RIA, we show that the human osteosarcoma cells synthesize significant amounts of BGP without any prior induction of BGP synthesis by 1,25-dihydroxyvitamin D. After specific immunoprecipitation of poly-A+ RNA in vitro translation products with antibodies to BGP, we found that BGP is synthesized as a precursor with an apparent mol wt of 13.5K, as demonstrated on 15% sodium dodecyl sulfate-polyacrylamide gels. Finally, pulse labeling of the osteosarcoma cells with [3H]proline reveals that the cells synthesize mature BGP of 12,000 mol wt as well as a higher mol wt precursor (13,500) of the protein.

Mutagen formation in a model beef boiling system III. purification and identification of three heterocyclic amine mutagens‐carcinogens

Abstract An extensively boiled supernatant2 (S2) fraction from beef round steak contains two major Salmonella frameshift mutagens, designated as HPLC peaks A and B. These same mutagens arise, but in different proportions, when S2 is boiled with creatine phosphate (CP), and they are produced in much greater quantities from a boiled mixture of S2 + L‐tryptophan (Trp) + CP + FeSO4 (S2 ∗). A third mutagen, peak C, is also generated in the S2 ∗ mixture. Mutagen peaks A, B, and C were purified to homogeneity and shown by their absorption spectra, mass fragmentation patterns, and other data to be 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 3‐amino‐l‐methyl‐5H‐pyrido[4,3‐b]‐indole (Trp‐P‐2), and 3‐amino‐l,4‐dimethyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐1), respectively. This is the first demonstration that IQ, Trp‐P‐2, and Trp‐P‐1 can form under aqueous conditions at 100°C from reactions between low molecular wt precursors that are present in meat juice. Further simplification of and studies with the S2 fraction of be...

Biosynthesis of the Epidermal Growth Factor Receptor in Cultured Human Cells*

A 160,000 mol wt precursor of the epidermal growth factor (EGF) receptor has been identified in human A-431 carcinoma cells and skin fibroblasts. The presence of one discrete precursor band indicates the presence of a slow processing step. We have determined that this slow processing step involves the conversion of high mannose N-linked oligosaccharides on the receptor precursor to primarily complex oligosaccharides on the mature form of the receptor. This is shown by 1) the presence of fucose, a characteristic terminal sugar of complex oligosaccharides, in only the mature receptor and by 2) the susceptibility of the precursor to digestion with endoglycosidase H, which cleaves high mannose N-linked oligosaccharides, but not complex oligosaccharides from glycoproteins. The precursor to mature receptor transition half-time is 1.7 h in A-431 cells. This long transition half-time causes an accumulation of approximately 7.2 X 10(5) precursor molecules per cell (approximately 12% of the total population of EGF receptors). The net quantity of mature EGF receptors, but not of receptor precursors, is reduced when EGF is added to the culture medium of A-431 cells. The presence of EGF in the growth medium also decreases electrophoretic migration (as a result of increased phosphate incorporation) of the mature receptor, but not that of the precursor. The EGF-insensitive state of the precursor is most likely due to its intracellular location.

Transport and proteolytic processing of rabbit cardiac cathepsin D.

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

Pancreatic somatostatinoma: abundance of somatostatin-28(1-12)-like immunoreactivity in tumor and plasma.

In the present study we characterized and compared the relative amounts of the different molecular forms of somatostatin-14 like immunoreactivity (S-14 LI) and of somatostatin-28(1-12) like immunoreactivity (S-28(1-12) LI) in extracts of tumor and peripheral plasma of a patient with a pancreatic somatostatinoma. Tissue and plasma were chromatographed on Sephadex G-50 columns equilibrated with 6 M urea. Immunoreactivity in the eluting fractions was assayed with two separate, region specific RIAs using antibodies R149 (S-14 LI) and S309 (S-28(1-12)LI). RIA R149 recognizes the 6-8 and 14 regions of the S-14 sequence and detects S-14, S-28, and prosomatostatin, an approximately 14,000 mol wt precursor for the two peptides. RIA S309 recognizes the 2-11 segment of S-28 and reacts with S-28, S-28(1-12), and higher mol wt S-28(1-12) LI but not S-14. Total tumor S-14 LI was 190 pmol/mg protein and consisted of three peaks of immunoreactivity of apparent 14,000 mol wt (14K S-14 LI), 3,200 mol wt (3.2K corresponding to S-28) and 1,600 mol wt (1.6K corresponding to S-14). The three peaks comprised, respectively, 7%, 57%, and 36% of total S-14 LI. Total tumor S-28(1-12) LI was 594 pmol/mg protein and eluted as four major peaks of immunoreactivity as follows: peak I (mol wt 15,000, 10% of total S-28(1-12) LI); peak II (mol wt 8,000, 20% of S-28(1-12) LI), peak III (corresponding to S-28, 19% of S-28(1-12) LI); peak IV (corresponding to S-28(1-12), approximately 50% of total S-28(1-12) LI). Total plasma concentration of S-14 LI was 714 pM, being made up of the three peaks found in tumor but in the following relative amounts (14K S-14 LI, 22%; 3.2K, 29%; 1.6 K, 49%). Plasma S-28(1-12) LI was 4 times higher (2879 pM) than S-14 LI and contained immunoreactivity corresponding to each of the four peaks found in the tumor. 1) The tumor and plasma concentrations of S-28(1-12) LI were greater than that of S-14 LI. 2) Both tumor and plasma S-14 LI and S-28 LI were heterogeneous and comprised species corresponding not only to S-14 but also S-28, S-28(1-12), prosomatostatin, and other higher mol wt forms of S-28.(ABSTRACT TRUNCATED AT 400 WORDS)

Surface antigens of malaria merozoites. A high molecular weight precursor is processed to an 83,000 mol wt form expressed on the surface of Plasmodium falciparum merozoites.

A technique was developed for obtaining high yields of naturally released Plasmodium falciparum merozoites from synchronous cultures of parasitized erythrocytes. The cultured erythrocytes were treated with trypsin to prevent reinvasion (6), and the released merozoites that accumulated extracellularly were harvested by differential centrifugation. The total biosynthetically labeled proteins of schizonts and merozoites, and those immunoprecipitated by human immune serum were analyzed and compared. The surface antigens of free merozoites, labeled by lactoperoxidase-catalyzed iodination, were also described. A monoclonal antibody, specific for a 195,000 mol wt schizont protein, and processing fragments derived from it (3) were used in immunoprecipitation and Western transfer analyses to determine which of the processing fragments are associated with merozoites and which of them are located on the merozoite surface. It was found that processing of the 195,000 mol wt precursor down to an 83,000 mol wt fragment is complete in free merozoites, and that this fragment is expressed as one of the major surface antigens of P. falciparum merozoites.