The length of the hydrophobic core of the bovine parathyroid hormone signal peptide was modified by in vitro mutagenesis. Extension of the hydrophobic core by three amino acids at the NH2-terminal end had little effect on the proteolytic processing of the signal peptide by microsomal membranes. Deletion of 6 of the 12 amino acids in the core eliminated translocation and processing of the modified protein. Deletion of pairs of amino acids across the core resulted in position-dependent inhibition of signal activity unrelated to hydrophobicity but inversely related to the hydrophobic moments of the modified cores. Deletions in the NH2-terminal region of the core were strongly inhibitory for proteolytic processing whereas deletions in the COOH-terminal region had no effect or increased processing when assessed either co-translationally with microsomal membranes or post-translationally with purified hen oviduct signal peptidase. Deletion of cysteine 18 and alanine 19 increased processing, but deletion of cysteine alone or substitution of leucine for cysteine did not increase processing more than deletion of both residues at 18 and 19. Translations of the translocation-defective mutants with pairs of amino acids deleted in a wheat germ system were inhibited by addition of exogenous signal recognition particle suggesting that interactions of the modified signal peptides with signal recognition particle were normal. The position-dependent effects of the hydrophobic core modifications indicate that structural properties of the core in addition to hydrophobicity are important for signal activity. The parallel effects of the modifications on co-translational translocation and post-translational processing by purified signal peptidase suggest that proteins in the signal peptidase complex might be part of, or intimately associated with, membrane proteins involved in the translocation. A model is proposed in which the NH2-terminal region of the hydrophobic core binds to one subunit of the signal peptidase while the other subunit catalyzes the cleavage.
Background: Bone grafts are used in approximately one half of all musculoskeletal surgeries. Autograft bone is the historic gold standard but is limited in supply and its harvest imparts significant morbidity to the patient. Alternative sources of bone graft include allografts, synthetics and, less commonly, xenografts which are taken from animal species. Xenografts are available in unlimited supply from healthy animal donors with controlled biology, avoiding the risk of human disease transmission, and may satisfy current demand for bone graft products. Methods: In the current study, cancellous bone was harvested from porcine femurs and subjected to a novel decellularization protocol to derive a bone scaffold. Results: The scaffold was devoid of donor cellular material on histology and DNA sampling (p < 0.01). Microarchitectural properties important for osteoconductive potential were preserved after decellularization as shown by high resolution imaging modalities. Proteomics data demonstrated similar profiles when comparing the porcine bone scaffold against commercially available human demineralized bone matrix approved for clinical use. Conclusion: We are unaware of any porcine-derived bone graft products currently used in orthopaedic surgery practice. Results from the current study suggest that porcine-derived bone scaffolds warrant further consideration to serve as a potential bone graft substitute.
To the Editors: We read with great interest the article by Hellstrom et al. (1) about characterization of soluble mesothelin proteins (SMP) in cancer patients. The authors found that the SMP in ascites from a patient with ovarian carcinoma contained the sequence of extracellular domain of a membrane-bound mesothelin (2, 3) rather than a soluble form speculated previously by Scholler et al. (4). Their results indicate that membrane-bound mesothelin could be shed from the tumor cells to generate SMP detected in patient sera by ELISA. We agree with their conclusion that membrane-bound mesothelin is the predominant protein present in the serum and here provides direct evidence showing that such shedding does in fact take place.Our experiments are summarized in Fig. 1. Using an SS1P affinity column, we purified SMP from culture supernatants of a well-documented A431/K5 cell line (3) expressing only membrane-bound mesothelin. SS1P is an immunotoxin with an Fv specific for mesothelin. About 100 ng/mL SMP was found in the culture supernatant. The molecular weight of purified SMP was ∼40 kDa (Fig. 1A), consistent with the SMP described in Hellstrom et al. (1). The full-length mesothelin precursor proteins were not detected in culture supernatant. After deglycosylation with PNGase, the molecular weight of SMP was ∼34 kDa. The SMP bands were cut out from a Coomassie-stained gel, protein in the gel was digested with trypsin, and the tryptic peptides were analyzed using liquid chromatography ion trap mass spectrometry (Fig. 1B). The sequence of NH2 terminal (EVEK) was determined by automated Edman degradation, and peptides corresponding to multiple internal regions of mesothelin were determined using the mass spectrometry/mass spectrometry mode on the ion trap mass spectrometer. Our results confirmed that the SMP was the extracellular domain of membrane-bound mesothelin shed from cells.Mesothelin is a glycosyl-phosphatidylinositol–anchored protein. Release of such proteins from the cell surface into the serum and other body fluids can be mediated by phospholipase or proteases (5). To determine whether the shedding of mesothelin is dependent on phosphatidylinositol-specific phospholipase C, we did anti-cross-reacting determinant assay (Prozyme, San Leandro, CA) by Western blot. The SMP was not recognized by anti-cross-reacting determinant (Anti-CRD) antibodies (Fig. 1C).In conclusion, our data show that the extracellular domain of membrane-bound mesothelin can be shed from tumor cells. The shedding may not require phosphatidylinositol-specific phospholipase C phospholipolysis. The precise mechanisms need further investigation.
BACKGROUND: WHO grade II low-grade gliomas (LGGs) with high risk factors for recurrence are mostly lethal despite current treatments, and novel approaches are needed. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in human leukocyte antigen (HLA)-A2+ adults with high-risk LGGs in the following three cohorts: 1) newly diagnosed patients without prior radiation therapy (RT); 2) newly diagnosed patients with prior RT, and 3) recurrent patients. METHODS: GAAs were interleukin-13 receptor (IL-13R)α2, EphA2, Wilms Tumor (WT)1, and Survivin, and synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for 8 courses with intramuscular injections of Toll-like receptor 3 agonist Polyinosinic-Polycytidylic Acid Stabilized by Lysine and Carboxymethylcellulose (poly-ICLC), followed by q12-week booster vaccines. Primary endpoints were safety and CD8+ T-cell responses against vaccine-targeted GAAs. RESULTS: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity has been encountered except for one case with Common Terminology Criteria for Adverse Events (CTCAE) Grade 3 fever (Cohort 1). Enzyme-linked Immuno-SPOT (ELISPOT) assays demonstrated robust and sustained interferon (IFN)-γ responses against at least 3 of the 4 GAA epitopes in 10 and 4 cases of Cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFN-γ ELISPOT responses than Cohort 3 patients, suggesting newly diagnosed patients have superior vaccine-responsiveness to recurrent patients. IFN-γ ELISPOT response levels in this study is significantly higher than those observed in our previous phase I/II study in high-grade glioma patients (Okada et al. JCO 2011). Furthermore, IFN-γ ELISPOT response levels were significantly higher than those for IL-5, indicating effective type-1 skewing by the current regimen. Median progression-free survival (PFS) periods are 21 months (Cohort 1; since diagnosis; range 10-42) and 12 months (Cohort 3; since the 1st vaccine; range 3-26). The only patient with large astrocytoma in Cohort 2 has been progression-free for over 58 months since diagnosis. There was a positive trend between IFN-γ ELISPOT responses and progression-free survival (PFS) in Cohort 3 patients (P = 0.08 by The Cox proportional hazards model). CONCLUSIONS: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade II LGG patients. These results suggest these patients may be suitable populations for vaccine therapy and warrant further evaluations of this approach. SECONDARY CATEGORY: Clinical Neuro-Oncology.
Type I signal peptidases cleave the signal peptides from secretory and membrane-associated proteins that are transported across or into the bacterial cell membrane or the lipid bilayer of the endoplasmic reticulum (ER) [1]. These peptidases are essential for life in most, if not all cells. In prokaryotes, they are single-chain integral membrane proteins that are anchored to the cell membrane by one or two membrane anchors. The eukaryotic enzyme is associated with a multi-subunit membrane protein complex with subunits positioned on both sides of the ER membrane. Current evidence suggests that the prokaryotic and eukaryotic signal peptidases are distantly related enzymes that use an atypical and perhaps very ancient proteolytic mechanism but few details are known about that mechanism. This chapter considers current information about the structure and functions of type I signal peptidases in prokaryotes and in the ER of eukaryotes. Related signal peptidases found in mitochondria [2] and chloroplasts [3] are not considered here.
Poly(A)‐containing RNA was isolated from chicken liver and translated in a reticulocyte lysate protein‐synthesizing system in the presence of radiolabeled amino acids. Chicken albumin was isolated from the translation products by immunoprecipitation, and subjected to automated Edman radiosequencing. Comparison with the sequence of proalbumin showed that the translation product (preproalbumin) contains an NH 2 ‐terminal extension of 18 amino acid residues. The NH 2 ‐terminal sequence of chicken preproalbumin was as follows: Met −18 ‐Lys‐Asn‐Val −15 ‐Thr‐Leu‐Ile‐Ser‐Phe −10 ‐Ile‐Phe‐Leu‐Phe‐Ser −5 ‐Ser‐Ala‐Thr‐Ser −1 ‐Arg 1 , where Arg 1 represents the NH 2 ‐terminal residue of proalbumin. This NH 2 ‐terminal extension is very rich in hydrophobic amino acid residues and is similar to the signal sequences found in other secreted proteins. The signal sequence of chicken preproalbumin shows considerable homology with the signal sequences of rat and bovine preproalbumins, but little homology with the signal sequences of other chicken preproteins.
