Antithrombin activity of the hirudin N-terminal core domain residues 1–43

Hirudin N-terminal core domain residues 1–43 (r-Hir^1–43) were prepared from limited proteolysis of recombinant hirudin by V8 Staphylococcal protease followed by purification with reversed-phase HPLC. r-Hir^1-43 lacks the putative reactive site of hirudin (Lys⁴⁷), but binds to thrombin (with K_i of 300 nM) and blocks the catalytic activity of the protease. The structural element which accounts for the thrombin inhibitory activity of r-Hir^1–43 is analyzed in this report.     

Characterization and purification of a protease in serum that cleaves proatrial natriuretic factor (ProANF) to its circulating forms

Atrial natriuretic factor (ANF) is synthesized and stored in atrial cardiocytes as a 17-kilodalton (kDa), 126 amino acid polypeptide, proANF, but circulates as smaller, 24 and 28 amino acid peptide fragments of the carboxy terminus of proANF. It has previously been shown that proANF is secreted intact from cultured atrial cardiocytes and can be cleaved by a serum protease to smaller, 3-kDa peptides believed to be the circulating forms. This report describes the purification and characterization of this proANF-cleaving protease from rat serum. The cleavages both of 35S-labeled proANF derived from rat atrial cell cultures, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/autoradiography, and of a synthetic p-nitroanilide-containing substrate were used as assays for the detection of enzyme activity. ProANF-cleaving activity was found in rat serum, with no such activity detectable in rat plasma. Cleavage in serum was not dependent on the presence of platelets or other cellular elements. Complete inhibition of proANF cleavage was obtained with the protease inhibitors benzamidine, leupeptin, phenylmethanesulfonyl fluoride, and diisopropyl fluorophosphate (DFP) but not with aprotinin, soybean trypsin inhibitor, pepstatin, or hirudin. Unlike the vitamin K dependent plasma proteins, the proANF-cleaving protease did not adsorb to barium sulfate. With the sequential application of ion-exchange, hydroxylapatite, lectin affinity, and gel filtration chromatography, a 5000-6000-fold purification of the enzyme from rat serum was achieved. Fractionation of either whole serum or the purified enzyme by gel filtration chromatography revealed a single peak of activity corresponding to a protein with a Stokes radius of 45 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH₂-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST_[1–10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys¹³ followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST_[1–10] production. Since the prosomatostatin sequence R⁸–Q⁹–F¹⁰–L¹¹↓ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST_[1–10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST_[1–10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST_[1–10] required the RXRXXL motif. However, this NH₂-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.     

Characterization of an endoprotease from rat small intestinal mucosal secretory granules which generates somatostatin-28 from prosomatostatin by cleavage after a single arginine residue

We have extracted, characterized, and partially purified an enzyme from secretory granules from rat small intestinal mucosa which cleaves a synthetic prosomatostatin substrate on the carboxyl side of a single arginine residue. This substrate Leu-Gln-Arg-Ser-Ala-Asn-Ser-NH2 contains the monobasic site at which mammalian prosomatostatin is cleaved in vivo to generate somatostatin-28. This activity was released from the granules by osmotic shock followed by extraction with 500 mM KCl. The enzyme had a molecular weight of about 55,000, a pH optimum of about 7.5, and a Km for the synthetic substrate of 20 microM. It was partially inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, iodoacetate, soybean trypsin inhibitor, and EDTA. It was also very sensitive to aprotinin (complete inhibition at 25 micrograms/ml) but was not inhibited by bestatin, pepstatin, or p-chloromercuribenzoate. This endoprotease was unable to cleave three small trypsin and kallikrein substrates (N alpha-benzoyl-L-arginine ethyl ester, N alpha-benzoyl-DL-arginine p-nitroanilide, and N alpha-benzoyl-L-arginine 7-amido-4-methylcoumarin). It was unable to cleave either the Arg-Asp bond in CCK 12 or the Arg-Glu and Arg-Met bonds of synthetic peptides corresponding to sequences of anglerfish prosomatostatin II situated upstream from the somatostatin-28 domain. These observations together suggest that adjacent amino acids play a role in determining the conformational specificity of the monobasic cleavage. This soluble enzyme was also able to cleave three synthetic substrates containing dibasic residues (Arg-Lys or Lys-Arg) on the carboxyl side of the arginine, although it did so less rapidly than at the monobasic cleavage sites. When incubated with partially purified prosomatostatin from anglerfish pancreas, significant quantities of somatostatin-28 II were produced. All these cleavages were completely blocked by preincubation with aprotinin. Although further work is required to clarify the physiological role of this enzyme, it appears, in view of its catalytic properties, this endoprotease could be involved in the conversion of prosomatostatin to somatostatin-28 in intestine mucosal secretory cells.     

Hydrolysis of fibrinogen and plasminogen by immobilized earthworm fibrinolytic enzyme II from Eisenia fetida

Earthworm fibrinolytic enzyme II (EFE-II) from Eisenia fetida has a broad hydrolytic specificity for peptide bonds. Our experiments show that EFE-II can hydrolyze the specific chromogenic substrates of thrombin (Chromozym TH), trypsin (Chromozym TRY) and elastase (Chromozym ELA). The Michaelis–Menten constant (K_m) for Chromozym ELA (245 μM) is much higher than those for the thrombin (90 μM) and trypsin (60 μM) substrates. On the other hand, EFE-II is inhibited most strongly by soybean trypsin inhibitor (SBTI), and weakly inhibited by elastinal, suggesting that EFE-II has a trypsin-like activity. Degradation of plasminogen (PLg) and fibrinogen by EFE-II was investigated after EFE-II had been immobilized onto 1,1′-carboryl-diimidazole (CDI)-activated Sepharose CL-6B. The immobilized EFE-II has 55–60% activity of the native enzyme with a higher thermal and pH resistance. EFE-II cleaves PLg at four hydrolytic sites: Lys₇₇–Arg₇₈, Arg₃₄₂–Met₃₄₃, Ala₄₄₄–Ala₄₄₅ and Arg₅₅₇–Ile₅₅₈. The site Arg₅₅₇–Ile₅₅₈ is also recognized and cleaved by tissue plasminogen activator (t-PA) and urokinase (UK), producing active plasmin. Cleaving Ala₄₄₄–Ala₄₄₅ released mini-plasmin with secondary activity to hydrolyze fibrin. Immobilized EFE-II degrades not only the A chain of fibrinogen in the C-terminal region (like human neutrophil elastase, HNE), but also in the N-terminal region at the Val₂₁–Glu₂₂ site.     

Degradation of human atrial natriuretic peptide by human brain membranes

Human atrial natriuretic peptide (Ser 99-Tyr 126) was rapidly degraded by both choroid plexus and hypothalamic membranes with a complex pattern of cleavage. The use of protease inhibitors allowed a preliminary characterization of the enzymes involved in the hydrolysis of the Ser-Phe and Phe-Arg bonds of iodine-labelled atrial natriuretic peptide.The C-terminal tripeptide was generated by three different enzymatic activities acting on the Ser-Phe bond: endopeptidase 24.11, a phosphoramidon-insensitive metallopeptidase and a thiol protease. Peptides like substance P, neurotensin, bradykinin inhibited the cleavage of the Ser-Phe bond of atrial natriuretic peptide. The C-terminal tripeptide was further degraded by aminopeptidases. Cleavage of the C-terminal dipeptide was inhibited by aprotinin, suggesting the contribution of brain kallikrein in the formation of this metabolite.These results show that many different proteases were involved in the hydrolysis of the C-terminal sequence of atrial natriuretic peptide, at least in vitro and underline the complexity of neuropeptide catabolism by brain preparations.     

Peptides from the N-terminus of the atrial natriuretic factor prohormone enhance guanylate cyclase activity and increase cyclic GMP levels in a wide variety of tissues

The 98 amino acid (a. a.) N-terminus of the 126 a. a. atrial natriuretic factor (ANF) prohormone contains three peptides consisting of a. a. 1–30 (proANF 1–30), a. a. 31–67 (proANF 31–67) and a. a. 79–98 (proANF 79–98) with blood pressure lowering, sodium and/or potassium excreting properties similar to atrial natriuretic factor (a. a. 99–126, C-terminus of prohormone). ProANF 1–30 and proANF 31–67 have separate and distinct receptors from ANF in both vasculature and in the kidney to help mediate the above effects. At the cellular level proANFs 1–30, 31–67, and 79–98 as well as ANF's effects are mediated by enhancement of the guanylate cyclase (EC 4.6.1.2) — cyclic GMP system in vasculature and in the kidney. These peptides from the N-terminus of the ANF prohormone circulate normally in man and in all animal species tested. The object of the present investigation was to determine if these peptides have the ability to enhance either guanylate cyclase and/or adenylate cyclase in a variety of other tissues in addition to kidney and vasculature. ProANF 1–30, proANF 31–67, proANF 79–98, and ANF all increased rat lung, liver, heart and testes, but not spleen, particulate guanylate cyclase 2- to 3-fold at their 100 nM concentrations. Dose response curves revealed that maximal stimulation of particulate guanylate cyclase activity by these newly discovered peptides was at their 1 M concentrations, with no further increase in activity above their 1 M concentrations. Half-maximal (EC₅₀) enhancement of particulate guanylate cyclase occurred at 0.15 ± 0.01, 0.3 ± 0.02, 0.5 ± 0.03, and 0.9 ± 0.03 nM for proANF 1–30, proANF 31–67, proANF 79–98 and ANF, respectively. ProANFs 1–30, 31–67, 79–98, and 99–126 (i.e., ANF) each increased cyclic GMP but not cyclic AMP levels in tissue slices of liver, lung, small intestine, heart, and testes. None of these peptides enhanced either adenylate cyclase or the soluble 100,000 G form of guanylate cyclase. The ability of these N-terminal peptides to enhance particulate guanylate cyclase activity in a wide variety of tissues suggests that they may have effects in a much wider variety of tissues than presently thought.     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

Distinct importin recognition properties of histones and chromatin assembly factors

The adhesive protein vitronectin (75 kDa) occurs in human blood fluid in a one-chain (Vn₇₅) or a two-chain form (Vn₆₅₊₁₀), and is produced by a specific cleavage (at Arg³⁷⁹–Ala³⁸⁰), by a proteinase not identified hitherto. These two forms were shown to be functionally different and therefore, this cleavage may have a regulatory significance in vivo. Here, we report the use of a tailored one-chain recombinant Vn, a specific protein kinase A phosphorylation at Ser³⁷⁸, and sequence analysis to show: (1) that none of the proteinases originating from blood, previously thought to be the endogenous proteinase (plasmin, thrombin, tPA, and uPA), is indeed the in vivo convertase; and (2) that furin, a serine endoproteinase residing in the secretory pathway of hepatocytes, where Vn is synthesized, specifically cleaves Vn at the endogenous cleavage site. Consequently, we propose that the Vn₇₅ to Vn₆₅₊₁₀ conversion takes place in the liver (not in blood) and is carried out by furin.     

Nardilysin cleaves peptides at monobasic sites

Nardilysin (N-arginine dibasic convertase, EC 3.4.24.61) was first identified on the basis of its ability to cleave peptides containing an arginine dibasic pair, i.e., Arg-Arg or Arg-Lys. However, it was observed that an aromatic residue adjacent to the dibasic pair (i.e., Phe-Arg-Lys) could alter the cleavage site. In this study we determined whether nardilysin can cleave peptides at a single basic residue. Nardilysin cleaves beta-endorphin at the monobasic site, Phe(17)-Lys(18), with a k(cat)/K(m) of 2 x 10(8) M(-)(1) min(-)(1). This can be compared to a k(cat)/K(m) of 8.5 x 10(8) M(-)(1) min(-)(1) for cleavage between a dibasic pair in dynorphin B-13. Nardilysin also cleaves calcitonin at His-Arg and somatostatin-14 at Cys-Lys. We examined the hydrolysis of fluorogenic peptides based on the beta-endorphin 12-24 sequence, Abz-T-P-L-V-T-L-X(1)-X(2)-N-A-I-I-K-Q-EDDnp. Nardilysin hydrolyzes the peptides when X(1)-X(2) = F-K, F-R, W-K, M-K, Y-K, and L-K. The kinetics of cleavage at F-K and F-R are similar; however, K-F is not hydrolyzed. Nardilysin cleaves at two monobasic sites M-K and F-R of the kallidin model peptide Abz-MISLMKRPPGFSPFRSSRI-NH(2), releasing desArg(10) kallidin (KRPPGFSPF). However, nardilysin does not release desArg(10) kallidin from the physiological precursor low molecular weight kininogen. These studies extend the range of potential substrates for nardilysin and further substantiate that nardilysin is a true peptidase.     

P-3A and (−)-desacetamido P-3A: demonstration and Study of their effective functional cleavage of duplex DNA

A study of the Fe(II) complexes of P-3A (1) and (−)-desacetamido P-3A (2) abilities to cleave duplex DNA was conducted through examination of single-strand and double-strand cleavage of supercoiled φX174 RFI DNA (Form I) in the presence of O₂ to produce relaxed (Form II) and linear (Form III) DNA, respectively. Like Fe(II)-bleomycin A₂ and deglycobleomycin A₂, Fe(II)-1 and 2 effectively produced both single- and double-strand cleavage of supercoiled φX174 DNA. Unlike Fe(II)-bleomycin A₂ or deglycobleomycin A₂, Fe(II)-1 and 2 were found to cleave duplex w794 DNA with no discemible sequence selectively suggesting that the polynucleotide recognition of the C-terminus tetrapeptide S subunit of the bleomycins including the bithiazole may dominate the bleomycin A₂ DNA cleavage selectivity.     

Characterization of C-terminal-truncated urinary metabolites of a recombinant hirudin in rats

Although it has been reported that hirudin was excreted in urine mainly as its nonmetabolized form in humans, dogs, and rabbits, no report has been published about the molecular nature of urinary metabolites in rats. We found that nonmetabolized hirudin could not be detected in rat urine after its i.v. administration and that urinary metabolites of recombinant hirudin CX-397 consisted of at least the following six C-terminal-truncated peptides: CX-397^1–49, CX-397^1–50, CX-397^1–51, CX-397^1–52, CX-397^1–54, and CX-397^1–55, in the ratio of roughly 11, 51, 3, 11, 19, and 5%, respectively. In conclusion, the urinary metabolism of recombinant hirudin in rats is different from that in humans, dogs, and rabbits, suggesting that the handling of hirudin in rat kidney is unique among them.     

Specific binding sites for prohormone atrial natriuretic peptides 1-30, 31-67 and 99-126

Two peptides with vasodilatory properties consisting of amino acids 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1-30), (31-67), and (99-126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1-30), (31-67), and (99-126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1-30), (31-67), and ANF were 2.57, 59.91 and 40 fmols/10(6) cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1-30), (31-67), and (99-126) (ANF). Each peptide bound to DDT1 MF-2 membranes between 10(-8) to 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7)M. These results suggest that proANF(1-30) and proANF(31-67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.     

Cleavage specificity of a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159–66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl–lysyl–bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.     

Prohormone Thiol Protease and Enkephalin Precursor Processing: Cleavage at Dibasic and Monobasic Sites

Production of active enkephalin peptides requires proteolytic processing of proenkephalin at dibasic Lys-Arg, Arg-Arg, and Lys-Lys sites, as well as cleavage at a monobasic arginine site. A novel “prohormone thiol protease” (PTP) has been demonstrated to be involved in enkephalin precursor processing. To find if PTP is capable of cleaving all the putative cleavage sites needed for proenkephalin processing, its ability to cleave the dibasic and the monobasic sites within the enkephalin-containing peptides, peptide E and BAM-22P (bovine adrenal medulla docosapeptide), was examined in this study. Cleavage products were separated by HPLC and subjected to microsequencing to determine their identity. PTP cleaved BAM-22P at the Lys-Arg site between the two basic residues. The Arg-Arg site of both peptide E and BAM-22P was cleaved at the NH₂-terminal side of the paired basic residues to generate [Met]-enkephalin. Furthermore, the monobasic arginine site was cleaved at its NH₂-terminal side by PTP. These findings, together with previous results showing PTP cleavage at the Lys-Lys site of peptide F, demonstrate that PTP possesses the necessary specificity for all the dibasic and monobasic cleavage sites required for proenkephalin processing. In addition, the unique specificity of PTP for cleavage at the NH₂-terminal side of arginine at dibasic or monobasic sites distinguishes it from many other putative prohormone processing enzymes, providing further evidence that PTP appears to be a novel prohormone processing enzyme.     

Huntingtin cleavage induced by thrombin in vitro

Huntingtin (Htt) mutation causes Huntington's disease.Sequence analysis of Htt revealed apossible thrombin cleavage site in the N-terminal region of Htt.In order to investigate if thrombin can eleaveHtt,we expressed the N-terminal fragment (1-969) of wild-type (wt) Htt (Htt 1-969) in MCF-7 cells andstudied its cleavage pattern by thrombin in vitro.An expression plasmid pcDNA3-Htt-18Q-969 was used totransfect MCF-cells and Htt 1-969 expression was confirmed with immunofluorescence.Cell lysates wereincubated with thrombin (1 U/ml, 10 U/ml,and 30 U/ml) for 1 h in the presence or absence of hirudin,athrombin inhibitor.Htt fragments were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and detected with anti-Htt antibodies. An Htt fragment with molecular mass of approximately80 kDa was produced after incubation with thrombin.The size of this Htt fragment was anticipated bymolecular mass generated from thrombin-mediated cleavage at the amino acid 183 in the Htt.Production ofan 80 kDa fragment was inhibited by hirudin. This study provides the first evidence that Htt is cleaved bythrombin in vitro at amino acid 183.If endogenous thrombin cleaves Htt in vivo,the physiological significanceof thrombin-mediated cleavage of Htt should be further investigated.     

Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes

Atrial natriuretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 35S-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiography. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease.     

Cellular peptide processing after a single arginyl residue. Studies on the common precursor for pancreatic polypeptide and pancreatic icosapeptide

The processing of the common precursor for pancreatic polypeptide and pancreatic icosapeptide was studied in primary cultures of endocrine cells isolated from the duodenal part of the canine pancreas. Biosynthetically labeled peptides were characterized by enzymatic digestion and radiosequencing and compared to a COOH-terminally extended form of the icosapeptide which was isolated from canine pancreas and also sequenced. It was substantiated that, in these cell cultures, processing can be studied at a classical dibasic site between the pancreatic polypeptide and the icosapeptide, and at a monobasic processing site between the icosapeptide and its COOH-terminal extension. Pulse-chase experiments showed that the monobasic cleavage occurs later than the dibasic one in the biosynthetic process; the monobasic site was apparently not cleaved before the prohormone had been processed at the dibasic site. The monobasic processing could also be distinguished from the dibasic cleavage mechanism as, in time, the cells gradually lost the ability to cleave at the monobasic site while the dibasic processing was unaffected. It is concluded that monobasic conversion, which is important in the activation of a series of hormones, neuropeptides, and growth factors, is a distinct cellular processing mechanism.     

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC^1–77) is partially processed in the intermediate lobe of the pituitary to N-POMC^1–49 and lys-γ₃ -melanotropin. Two pools of N-POMC^1–77 were isolated which were differentially glycosylated at threonine⁴⁵, while N-POMC^1–49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC^1–77 may regulate cleavage at the Arg⁴⁹-Lys⁵⁰ processing site. We tested this hypothesis by incubating N-POMC^1–77 glycoforms with purified pro-opiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC^1–77 and O-glycosylated N-POMC^1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-γ₃ -melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.     

Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of beta-arrestins

β-Arrestins play a role in AT₁ endocytosis by binding the cytoplasmic, C-terminus region T332–S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting β-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT₁ to investigate two possibilities: activated AT₁ induces global relocation of β-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract β-arrestins. Results obtained using high osmolarity and dominant-negative β-arrestin confirmed that internalization of AT₁ in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving β-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT₁ internalization. Confocal microscopy revealed that wild-type AT₁ induced a time-dependent translocation of GFP-tagged β-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic β-arrestin 1 at all, and only trafficked β-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both β-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and β-arrestin 1 less so than β-arrestin 2. In conclusion, this study shows that the high affinity binding of β-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced β-arrestin recruitment to the cell surface and AT₁ receptor.