Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein.

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.     

Avian reticuloendotheliosis virus-transformed lymphoid cells contain multiple pp59v-rel complexes.

The v-rel oncogene of avian reticuloendotheliosis virus type T (REV-T) encodes a 59-kilodalton (kDa) phosphoprotein located principally in the cytosol of transformed lymphoid cells. All of the detectable pp59v-rel was present in high-molecular-weight complexes containing at least five cellular proteins (p124, p115, p75c-rel, p70hsc, and pp40). Antiserum was developed against the 40-kDa protein, the most abundant cellular protein associated with the complex. The 40-kDa phosphoprotein was complexed with pp59v-rel in REV-T-transformed lymphoid cell lines arrested at different stages of B-cell development as well as in lymphoid tumor cells and in fibrosarcomas. The half-life (8 h) of pp40 in REV-T-transformed lymphoid cells was the same as that of pp59v-rel. Antiserum against pp40 permitted the identification of two pp59v-rel complexes. The most abundant cytoplasmic complex contained approximately 75% of the pp59v-rel and all of the detectable pp40 in REV-T-transformed lymphoid cells. Twenty-five percent of the pp59v-rel was present in a minor complex that contained the majority of p75c-rel along with p115 and p124. In nuclear extracts of REV-T-transformed lymphoid cells, pp59v-rel was complexed with pp40. The two high-molecular-weight proteins (p115 and p124) and p75c-rel were not detected in the nuclear complex. In the cytosolic complexes, pp40 was heavily phosphorylated, whereas the nuclear form was much less extensively phosphorylated.     

Localization of the v-rel protein in reticuloendotheliosis virus strain T-transformed lymphoid cells.

The protein (p59rel) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel (N. R. Rice, T. D. Copeland, S. Simek, S. Oroszlan, and R. V. Gilden, Virology 149:217-229, 1986). To obtain polyclonal antibodies directed against a larger number of p59rel epitopes, a 262-amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-delta-rel) precipitated p59rel from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with the peptide antisera. We used this new antiserum to localize p59rel in REV-T-transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59rel was found in the cytosolic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies (Rice et al., Virology 149:217-229, 1986) it was shown that immune precipitates formed with one of the three p59rel peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59rel or a protein closely associated with it.     

Multiple forms of recombinant murine interleukin-4 expressed in COS-7 monkey kidney cells

Recombinant murine interleukin-4 (muIL-4) expressed in COS-7 monkey kidney cells was purified to homogeneity by sequential CM-Sepharose, Sephadex G-100 chromatography and mono-S FPLC to a specific activity of 6.10(7) units per mg of protein based on an in vitro HT-2 cell proliferation assay. Two electrophoretic variants, designated a and b, which migrated on SDS-PAGE as a closely spaced doublet with Mr 19,000, were present in the final product. Gas phase sequencing of the purified protein revealed the presence of an N-terminus corresponding to the mature protein predicted from the cDNA sequence and sequencing of a cyanogen bromide digest confirmed 75 of the 120 predicted amino acids. Elution behavior on gel filtration corresponded to that of a monomer of Mr 19,000. Since there are three potential sites of N-glycosylation predicted by the cDNA sequence, the contribution of glycosylation to the observed heterogeneity was examined by treatment with endoglycosidases. Variant b was digested by either endo-beta-N-acetylglucosaminidase H (endo H) or endo-beta-N-acetylglucosaminidase F (endo F) to protein of Mr 15,000 on SDS-PAGE but was unaffected by treatment with endo-beta-N-acetylglucosaminidase D (endo D), thus indicating the presence of high mannose type of N-glycan. In contrast, variant a was resistant to endo H, F and D. Complete conversion of a mixture of variants a and b to a single protein of Mr 15,000 on SDS-PAGE was obtained only after treatment with N-glycanase. Both variants were resistant to neuraminidase and O-glycanase treatment. These data show that the microheterogeneity observed in purified muIL-4 preparations is due to differences in the nature of the N-linked oligosaccharides. The availability of purified recombinant muIL-4 and a methodology for both total and selective deglycosylation provides a basis for the initiation of structure-function studies of this novel T-cell lymphokine.     

A 15,000-Mr protein proteolytically derived from vitellogenin within oocyte of Perinereis cultrifera (polychaete annelid). Identification, purification and partial characterization

It has been shown previously in our laboratory that, in Perinereis cultrifera, the four mature vitellin subunits (Mr 98,000, 22,000, 20,000, 16,000) are proteolytically derived within the oocyte from a single extraoocytic precursor, vitellogenin, with an apparent Mr (176,000) higher by 20,000 than the sum of the Mr of the four end products. In this report, it is shown that a 15,000-Mr protein, designated as P15, not only accumulates in maturing oocytes but also originates from outside these cells similarly to vitellin. Moreover in vivo labelling experiments indicate that the appearance of P15 occurs after vitellogenin enters the oocyte, concurrently with the appearance of the lower-Mr fragments characteristic of vitellin. From these data, it is concluded that P15 most likely represents a vitellogenin-derived protein which is generated within the oocytes during the transformation of vitellogenin into vitellin. This conclusion is further supported by the additional finding that P15 immunologically cross-reacts with vitellogenin but not with mature vitellin. P15 has been purified to homogeneity from the soluble protein fraction of submature oocytes and partially characterized. The 15,000-Mr protein exists in a monomeric form with a pI of about 7.7. Unlike vitellin, P15 does not contain significant amounts of lipid or carbohydrate and has a low absorbance at 280 nm. The amino acid composition of the purified protein is also presented.     

p15, A nuclear-associated protein of human sperm: Identification of four variants and their occurrence in normal and abnormal seminal cells

A basic protein of apparent molecular weight 15,000 d (p15) has been identified as a tissue-specific species-unique component of spermatogenic cells of human semen. Cytoimmunochemical study with a monoclonal antibody indicates that p15 resides in a perinuclear space in morphologically normal spermatozoa and differs in distribution and stat in abnormal seminal cell and nonnucleated bodeis. Biochemical analysis indicateds that p15 occurs as four variiants, differentially migratory on acetic acid/urea gel and differnetially extractable by NaCl in reducing solution. By correlation of the cytologic and biochemical data, we propose that variant 1 is the unmodified form of p15; in the normal progression of spermiogenesis p15 is modified to variants 2 and 3 and in the absence of the normal progression is unmodified or aberrantly modified to variant 4. The association of molecular abnormality in p15 with morphologically abnormal sperm suggests that p15 may play a role in sperm-head shaping.     

Correlation between the release of ovine trophoblast protein-1 by the conceptus and the production of polypeptides by the maternal endometrium of ewes

We studied the biosynthesis of two proteins, p70 (Mr 70,000; pI 4.0) and p15 (Mr 15,000; pI 5.7), by endometrial tissues from ewes between Days 12 and 24 of pregnancy and between Days 12 and 16 of the oestrous cycle to determine whether production of the two was correlated with the period of biosynthesis of ovine trophoblast protein-1 (oTP-1) by the conceptus. We also compared the protein synthetic activities of endometrium from gravid and non-gravid horns of pregnant ewes at Days 14, 16 and 18 in which the conceptus had been confined to one uterine horn. Proteins p70 and p15 were produced maximally between Days 14 and 20 of pregnancy, but synthesis by endometrial cultures from cyclic ewes was low or absent. Furthermore, synthesis of Protein p70 in particular was much greater by the gravid than non-gravid horn of unilaterally pregnant ewes. We conclude that synthesis of Proteins p70 and p15 by the uterus of sheep coincides with the time of oTP-1 production by the conceptus.     

Purification and properties of a megakaryocyte stimulatory factor present both in the serum-free conditioned medium of human embryonic kidney cells and in thrombocytopenic plasma

Megakaryocyte stimulatory factor (MSF) has been purified to homogeneity (7.5 X 10(5)-fold) from serum-free conditioned medium obtained from cultured human embryonic kidney cells and to near homogeneity (1.44 X 10(7)-fold) from thrombocytopenic rabbit plasma. MSF activity from either source was assayed by its ability to enhance the rate of synthesis of platelet factor 4-like proteins in a rat promegakaryoblast cell line. The 125I-labeled factor prepared from human embryonic kidney cell conditioned medium is homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing in the presence of 9.2 M urea. MSF obtained from the above source is an acidic protein (pI = 5.1) with an Mr = 15,000 which stimulates platelet factor 4-like protein synthesis in rat promegakaryoblast cells by as much as 7-fold, and exhibits half-maximal activity at a concentration of 0.8 pM. MSF was also purified from thrombocytopenic rabbit plasma by a nearly identical isolation procedure, and 125I-labeled factor prepared from this source also possessed an Mr = 15,000. MSF exhibited no biologic activity corresponding to other known hemopoietic growth factors, and appears to be specific for the megakaryocyte lineage.     

Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary

Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts.     

The identification of O-glycosylated precursors of insulin-like growth factor II.

A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.     

Isolation and characterization of multiple variants of recombinant human interleukin 4 expressed in mammalian cells

We have purified recombinant human interleukin 4 (huIL-4), formerly named B-cell stimulatory factor-1, from supernatants of COS-7 monkey kidney and L-929 cells transfected with the cDNA for huIL-4. The purified protein exhibited a specific activity of 2.6 X 10(7) units/mg in a T-cell proliferation assay and consisted of multiple components on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibiting Mr values of 15,000, 18,000, and 19,000. All forms of huIL-4 eluted on gel filtration chromatography with an apparent Mr of 22,000. Gas-phase microsequencing identified 26 and 8 amino acid residues at the N and C termini, respectively, all of which were consistent with the cDNA sequence. The site of processing of the signal sequence was found to occur between Gly-24 and His-25. Incubation with N-glycanase converted the 18- and 19-kDa variants to a 15-kDa form. Treatment with endo-beta-N-acetylglucosaminidase H reduced the molecular mass of the 18-kDa variant to 15 kDa, but did not have any apparent effects on the mass of the 19-kDa species. The removal of oligosaccharide by any of these treatments did not affect bioactivity in the T-cell proliferation assay. Neither O-glycanase nor endo-beta-N-acetylglucosaminidase D affected the molecular weight of any of these species. These data suggest that differences in carbohydrate structure account, at least in part, for the observed microheterogeneity.     

Uterine-specific proteins in luminal secretions of swine leukocyte antigen-inbred miniature swine with cystic endometrial hyperplasia

Uterine-specific proteins were evaluated in luminal secretions of Swine Leukocyte Antigen (SLA)-inbred miniature swine with cystic endometrial hyperplasia (CEH) by polyacrylamide gel electrophoresis (PAGE) and Sephacryl S-200 chromatography. CEH and non-CEH (NCEH) pigs (n = 23) were killed on Days 4, 9, and 15 of the estrous cycle (estrus = Day 0) and reproductive tracts were excised for collection of serum and uterine luminal protein. Uterine luminal protein was greater (p less than 0.05) on Day 9 than on Days 4 and 15 (42.9 vs. 6.1 and 29.4 mg, respectively) for CEH pigs and Days 4, 9, and 15 (8.5, 10.1, and 25.6 mg, respectively) for NCEH pigs. The presence of the uterine-specific acidic and basic proteins, as revealed by PAGE, was affected (p less than 0.025) by day of the cycle and CEH condition. All Day 15 NCEH pigs (4 of 4) produced the complete profile of these proteins, whereas none of the uterine protein samples representing other treatment groups contained them. Some minor acidic protein components were present in cystic fluids from CEH pigs, but these fluids lacked the typical uterine-specific proteins. PAGE analysis of Sephacryl S-200 fractions from uterine fluids of Day 15 NCEH pigs revealed the uterine-specific proteins in fractions IV (Mr 40,000) and V (Mr 15,000). The results of the investigation demonstrate an impairment in the secretion of uterine-specific proteins in cyclic SLA miniature swine with cystic endometrial hyperplasia.     

Glycosylation is essential for efficient secretion but not for permeability-enhancing activity of vascular permeability factor (vascular endothelial growth factor)

The hyperpermeability of the microvasculature supplying solid tumors is largely attributable to a heterodimeric Mr 34,000-43,000 tumor-secreted protein, vascular permeability factor. Upon reduction, the vascular permeability factor secreted by line 10 tumor cells is resolved by SDS-PAGE into 3 discrete bands of Mr 24,000, 19,500, and 15,000. We demonstrate here that line 10 vascular permeability factor is an N-linked glycoprotein. Nonglycosylated vascular permeability factor migrates on reduced SDS-PAGE as two bands of Mr 20,000 and 15,000. Pulse-chase studies demonstrated that all three chains of native vascular permeability factor were secreted rapidly following synthesis and at equal rates, with a cellular half-retention time of approximately 37 min. When glycosylation was prevented by tunicamycin, individual bands of nonglycosylated vascular permeability factor were also secreted at equivalent rates, but much more slowly (approximately 60 min) than native glycoprotein. Both glycosylated and nonglycosylated forms of vascular permeability factor were equally potent at increasing dermal vessel permeability.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.