N-methyl-d, l-aspartate stimulates growth hormone but not luteinizing hormone secretion in the sheep

The objective of this experiment was to determine the effects of N-methyl-d, l-aspartate (NMA) on luteinizing hormone (LH) and growth hormone (GH) secretion in castrated male sheep. Blood was sampled from Hampshire wethers every 15 min for 8 hr on day 1. At 4 and 6 hr after the initiation of the experiment, wethers were treated i.v. with NMA at a dose of 12 mg/kg body weight (n = 5) or .9% saline (n = 5). The dosage of NMA was within the range of doses that was previously demonstrated to stimulate LH secretion in monkeys. Blood samples were also collected every 15 min for 1 hr on day 2, beginning 24 hr after the first injection of NMA or saline. Treatment with NMA had no effect on mean LH concentrations, LH pulse frequency or LH pulse amplitude during the 4 hr period following the first injection on day 1. On day 2, however, mean LH concentrations were lower (p less than .01) in NMA versus saline-treated wethers. Conversely, administration of NMA evoked a dramatic increase (p less than .02) in mean GH concentrations on day 1. The mechanisms responsible for the effects of NMA described herein and whether or not these effects are relevant to the physiological control of LH and GH release in the sheep warrants further scrutiny.     

Properties of a cleaved two-chain form of recombinant human growth hormone

Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.     

Production of a biologically active variant form of recombinant human secretin

A biologically active variant form of recombinant human secretin was produced using a gene fusion system designed to facilitate the purification of the protein. The fusion protein was recovered from the culture medium of Escherichia coli by IgG affinity chromatography, and recombinant secretin was released by cyanogen bromide treatment. A novel approach involving addition of a C-terminal Gly-Lys-Arg extension, was used to overcome the lack of amidation of recombinant proteins in Escherichia coli. The biological activity of the recombinant variant of secretin was at least 80% of the porcine secretin standard.     

Recovery of functionally active recombinant human phospholipid scramblase 1 from inclusion bodies using N-lauroyl sarcosine

Human phospholipid scramblase (hPLSCR1) is a transmembrane protein involved in rapid bidirectional scrambling of phospholipids across the plasma membrane in response to elevated intracellular calcium (Ca(2+)) levels. Overexpression of recombinant hPLSCR1 in Escherichia coli BL21 (DE3) leads to its deposition in inclusion bodies (IBs). N-lauroyl sarcosine was used to solubilize IBs and to recover functionally active hPLSCR1 from them. Protein was purified to homogeneity by nickel-nitrilotriacetic acid (Ni(2+)-NTA) affinity chromatography and was >98% pure. Functional activity of the purified protein was validated by in vitro reconstitution studies, ~18% of 7-nitrobenz-2-oxa-1, 3-diazol-4-yl-phosphatidylcholine (NBD-PC) phospholipids was translocated across the lipid bilayer in the presence of Ca(2+) ions. Far ultraviolet circular dichroism (UV-CD) studies reveal that the secondary structure of protein is predominantly an α-helix, and under nondenaturing conditions, the protein exists as a monomer. Here we describe a method to purify recombinant membrane protein with higher yield than previously described methods involving renaturation techniques.     

Two contrary functions of tenascin: dissection of the active sites by recombinant tenascin fragments

A structural and functional model of tenascin was elaborated using recombinant parts of three alternatively spliced tenascin variants and anti-tenascin monoclonal antibodies. The fusion proteins were compared with intact tenascin for their functions and by electron microscopy. A strong cell binding site was localized within 104 amino acids. This fragment also contains the epitope of the monoclonal antibody anti-Tn68, which inhibits cell attachment to tenascin and binds near the tips of the six arms of tenascin. In contrast, constructs containing the 13 1/2 EGF-like repeats of tenascin showed an antiadhesive effect. The coexistence of the two contrary signals on the same molecule might be responsible for the versatile features of tenascin.     

A membrane-anchored form but not the secretory form of human chorionic gonadotropin-alpha chain acquires polylactosaminoglycan

Large polylactosaminoglycans have only been observed linked to membrane proteins. To determine if membrane anchoring of a secretory protein might lead to the addition of polylactosaminoglycan, we have examined the carbohydrate structure on a membrane-anchored form of human chorionic gonadotropin-alpha subunit. This protein was generated by fusing the DNA encoding the human chorionic gonadotropin-alpha subunit to the DNA encoding the membrane-spanning and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. DNAs encoding this hybrid form and the secretory form of human chorionic gonadotropin-alpha were expressed in monkey COS-1 cells using an SV40-based vector. We show here that the parent secretory glycoprotein contains typical Asn-linked complex-type oligosaccharides while the membrane-bound form contains large, heterogenous polylactosaminoglycans. We conclude that membrane anchoring increases the accessibility of the N-linked glycans to the enzymes involved in polylactosamine addition. The inhibitor 1-deoxymannojirimycin blocks addition of the polylactosaminoglycan.     

HU and IHF, two homologous histone-like proteins of Escherichia coli, form different protein-DNA complexes with short DNA fragments.

Using the gel retardation technique we have studied the protein-DNA complexes formed between HU--the major histone-like protein of Escherichia coli--and short DNA fragments. We show that several HU heterodimers bind DNA in a regularly spaced fashion with each heterodimer occupying about 9 base pairs. The alpha 2 and beta 2 HU homodimers form the same structure as the alpha beta heterodimer on double stranded DNA. However when compared to the heterodimer, they bind single stranded DNA with higher affinity. We also show that HU and the Integration Host Factor of E. coli (IHF) form different structures with the same DNA fragments. Moreover, HU seems to enhance the DNA-binding capacity of IHF to a DNA fragment which does not contain its consensus sequence.     

Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c.

A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).     

Defective interfering particles of Sindbis virus do not interfere with the homologous virus obtained from persistently infected BHK cells but do interfere with Semliki Forest virus.

Defective interfering particles derived from wild-type Sindbis virus no longer interfere with the infectious virus cloned from BHK cells persistently infected with Sindbis virus for 16 months. These particles do interfere with the replication of Semliki Forest virus.     

Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation

Key message

Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent.

Abstract

Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein’s N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Production of biologically active forms of recombinant hepcidin, the iron-regulatory hormone

Hepcidin is a liver produced cysteine-rich peptide hormone that acts as the central regulator of body iron metabolism. Hepcidin is synthesized under the form of a precursor, prohepcidin, which is processed to produce the biologically active mature 25 amino acid peptide. This peptide is secreted and acts by controlling the concentration of the membrane iron exporter ferroportin on intestinal enterocytes and macrophages. Hepcidin binds to ferroportin, inducing its internalization and degradation, thus regulating the export of iron from cells to plasma. The aim of the present study was to develop a novel method to produce human and mouse recombinant hepcidins, and to compare their biological activity towards their natural receptor ferroportin. Hepcidins were expressed in Escherichia coli as thioredoxin fusion proteins. The corresponding peptides, purified after cleavage from thioredoxin, were properly folded and contained the expected four-disulfide bridges without the need of any renaturation or oxidation steps. Human and mouse hepcidins were found to be biologically active, promoting ferroportin degradation in macrophages. Importantly, biologically inactive aggregated forms of hepcidin were observed depending on purification and storage conditions, but such forms were unrelated to disulfide bridge formation.     

Expression and characterization of a soluble, active form of the jaagsiekte sheep retrovirus receptor, Hyal2

Retrovirus entry into cells is mediated by specific interactions between virus envelope glycoproteins and cell surface receptors. Many of these receptors contain multiple membrane-spanning regions, making their purification and study difficult. The jaagsiekte sheep retrovirus (JSRV) receptor, hyaluronidase 2 (Hyal2), is a glycosylphosphatidylinositol (GPI)-anchored molecule containing no peptide transmembrane regions, making it an attractive candidate for study of retrovirus entry. Further, the hyaluronidase activity reported for human Hyal2, combined with its broad expression pattern, may point to a critical function of Hyal2 in the turnover of hyaluronan, a major extracellular matrix component. Here we describe the properties of a soluble form of human Hyal2 (sHyal2) purified from a baculoviral expression system. sHyal2 is a 54-kDa monomer with weak hyaluronidase activity compared to that of the known hyaluronidase Spam1. In contrast to a previous report indicating that Hyal2 cleaved hyaluronan to a limit product of 20 kDa and was active only at acidic pH, we find that sHyal2 is capable of further degradation of hyaluronan and is active over a broad pH range, consistent with Hyal2 being active at the cell surface where it is normally localized. Interaction of sHyal2 with the JSRV envelope glycoprotein was analyzed by viral inhibition assays, showing >90% inhibition of transduction at 28 nM sHyal2, and by surface plasmon resonance, revealing a remarkably tight specific interaction with a dissociation constant (K_D) of 32 ± 1 pM. In contrast to results obtained with avian retroviruses, purified receptor was not capable of promoting transduction of cells that do not express the virus receptor.     

Human recombinant stem cell factor promotes spermatogonial proliferation,but not meiosis initiation in organ culture of newt testis fragments

We previously showed that mammalian FSH stimulates the proliferation of newt spermatogonia and induces their differentiation into primary spermatocytes in vitro. In the current study, to examine a possibility that stem cell factor (SCF) is involved in the proliferation of newt spermatogonia and/or their differentiation into primary spermatocytes, human recombinant SCF (rhSCF) was added to organ culture of testicular fragments. rhSCF was found to stimulate the spermatogonial proliferation and the spermatogonia progressed to the seventh generation that is the penultimate stage before primary spermatocyte stage. However, the spermatogonia did not differentiate into primary spermatocytes, but instead died of apoptosis. These results indicate that rhSCF promotes the proliferation of newt spermatogonia, but not the initiation of meiosis.     

Alternative splicing of the human Rab6A gene generates two close but functionally different isoforms

Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A', which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A' (Rab6A' Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A' Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A' is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A' interacts with two Rab6A partners, GAPCenA and "clone 1," but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A' are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.     

Purification and characterization of two active derivatives of recombinant YplA, a secreted phospholipase from Yersinia entercolitica

The virulence-associated phospholipase of Yersinia enterocolitica (YplA), which is secreted by a flagellar type III secretion system, was cloned and purified for structure-function analysis using a His(6)-tag expression system. Two versions of YplA have been proposed on the basis of two potential initiating methionine residues. The longer derivative possesses 59 additional amino acids at its N-terminus and appears to represent the native form of YplA; however, the shorter recombinant protein possesses enhanced activity in vitro. Both recombinant YplA derivatives are highly active as type-A(2) phospholipases and possess similar physical properties. Based on type III secretion substrates from other gram-negative bacteria, the N-terminus of YplA is probably required as a secretion signal; however, differences in the time-based activity of these two recombinant enzymes, the N-terminus of YplA may also have a regulatory function.     

Two subunits of glycosylphosphatidylinositol transamidase,GPI8 and PIG-T,form a functionally important intermolecular disulfide bridge

Many eukaryotic proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI transamidase is localized in the endoplasmic reticulum and mediates post-translational transfer of preformed GPI to proteins bearing a carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is a multimeric complex consisting of at least five subunits. Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues. GPI8 and PIG-T mutants in which relevant cysteines were replaced with serines were unable to fully restore the surface expression of GPI-anchored proteins upon transfection into their respective mutant cells. Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity. Antibodies against GPI8 and PIG-T revealed that endogenous as well as exogenous proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a similar intermolecular disulfide bond via its conserved cysteine residue, suggesting that the trypanosome GPI transamidase is also a multimeric complex likely containing the orthologue of PIG-T. We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins.     

Expression and purification of a functionally active recombinant GDP-mannosyltransferase (PimA) from Mycobacterium tuberculosis H37Rv

Lipoarabinomannans (LAM), especially mannose-capped LAM, abundant in the cell wall of Mycobacterium tuberculosis (Mtb) exhibit a broad spectrum of immunomodulatory functions and emerge as key virulence factors that may be relevant drug targets. The pimA gene of mycobacteria encodes a alpha-mannosyltransferase involved in the transfer reaction of the very first mannose from GDP-mannose to the carrier lipid phosphatidyl-myo-inositol, a precursor in the synthesis of LAM. PimA has been proposed to play an essential role in the growth of mycobacteria. In this study, the pimA gene from M. tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the Mtb PimA in fusion with a histidine-rich peptide on the N-terminal. The Mtb PimA was purified from the supernatant of the lysed cells under native conditions by immobilized metal affinity chromatography. The purity and molecular weight of Mtb PimA were determined by high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight. Circular dichroism spectroscopy study on Mtb PimA showed that the protein was folded. The enzyme assays revealed that Mtb PimA showed a requirement for Mg(2+) for the activity and the K(m) and V(max) values of Mtb PimA were estimated at 18 +/- 2 microM and 0.1 +/- 0.05 nmol/min/microg, respectively. This is the first report describing cloning and expression of GDP-mannosyltransferase gene of M. tuberculosis in E. coli.