Characterization of free alpha- and beta-chains of recombinant macrophage-stimulating protein

Human serum macrophage-stimulating protein (MSP) induces motile activity of murine resident peritoneal macrophages and is a growth and motility factor for epithelial cells. It belongs to the plasminogen-related family of kringle proteins, and is secreted as a single-chain, 78-kDa, biologically inactive pro-MSP. Proteolytic cleavage of pro-MSP at a single site yields active MSP, a disulfide-linked alphabeta-chain heterodimer. However cleavage of recombinant pro-MSP yielded not only the disulfide-linked heterodimer, but also free alpha- and beta-chains, indicating that some of the recombinant molecules lacked an alphabeta-chain disulfide. We purified the free chains for characterization. The beta-chain of MSP has three extra cysteines, Cys527, Cys562, and Cys672, which are not found in the plasminogen beta-chain. Disulfide bond analysis showed a Cys527-Cys562, but also a Cys588-Cys672. Coopting Cys588 by Cys672 prevented the expected formation of a disulfide between alpha-chain Cys468 and beta-chain Cys588. Concomitant studies determined structures of oligosaccharides at the three Asn-linked glycosylation sites of MSP. The oligosaccharides at the three Asn loci are heterogeneous; 11 different sugars were identified, all being sialylated fucosyl biantennary structures. We also located the pro-MSP signal peptide cleavage site at Gly18-Gln19 and the scissile bond for formation of mature MSP at Arg483-Val484.     

Secretion of Plasminogen Activator Inhibitor 2 by Human Peripheral Blood Monocytes Occurs via an Endoplasmic Reticulum–Golgi-Independent Pathway

Plasminogen activator inhibitor 2 (PAI-2) is a serine protease inhibitor (serpin) that is secreted and accumulated intracellularly by monocytes. We investigated PAI-2 synthesis by isolated human peripheral blood monocytes and found that a 47-kDa nonglycosylated form of PAI-2 was abundant in conditioned medium from monocytes. Secretion of PAI-2 by monocytes was not inhibited by agents that inhibit either ER–Golgi pathway-dependent secretion, brefeldin A, or N-linked glycosylation, tunicamycin. IL-1β served as a control for a protein that is secreted by an ER–Golgi-independent pathway, and secretion of IL-1β was not inhibited by brefeldin A. This was in contrast to secretion of TNFα, which was dependent on the ER–Golgi pathway. None of the treatments was cytotoxic toward monocytes, as measured by release of the intracellular enzyme lactate dehydrogenase (LDH) into the conditioned medium. Subcellular fractionation revealed that PAI-2 and IL-1β were colocalized. The mechanism for secretion of PAI-2 was not dependent on calcium or intracellular trafficking via the classical vesicular mechanism(s), distinguishing it from IL-1β secretion. These studies show that PAI-2 is secreted by primary human monocytes via an ER–Golgi-independent pathway.     

Biosynthesis and secretion of M- and Z-type alpha 1-proteinase inhibitor by human monocytes. Effect of inhibitors of glycosylation and of oligosaccharide processing on secretion and function

The biosynthesis and secretion of M-type and Z-type alpha 1-antitrypsin was studied in human monocytes. In monocytes of PiMM individuals alpha 1-antitrypsin represented 0.08% of the newly synthesized proteins and 0.44% of the secreted proteins. Two molecular forms of alpha 1-antitrypsin could be identified: a 51-kDa intracellular form, susceptible to endoglucosaminidase H, thus representing the high-mannose type precursor form and a 56-kDa form resistant to endoglucosaminidase H which was secreted into the medium. Inhibition of de novo glycosylation by tunicamycin impaired the secretion of M-type alpha 1-antitrypsin by about 75% whereas inhibition of oligosaccharide processing by the mannosidase II inhibitor swainsonine did not alter the secretion of M-type alpha 1-antitrypsin. alpha 1-Antitrypsin secreted by human monocytes was functionally active as measured by complex formation with porcine pancreatic elastase. Even unglycosylated alpha 1-antitrypsin secreted by human monocytes treated with tunicamycin formed a complex with elastase. In monocytes of PiZZ individuals the secretion of alpha 1-antitrypsin was decreased. 72% of newly synthesized M-type alpha 1-antitrypsin, but only 35% of newly synthesized Z-type alpha 1-antitrypsin were secreted during a labeling period of 3 h with [35S]methionine. The 51-kDa form of Z-type alpha 1-antitrypsin accumulated intracellularly, whereas the 56-kDa form was secreted. Inhibition of oligosaccharide processing by swainsonine did not alter the decreased secretion of Z-type alpha 1-antitrypsin, whereas inhibition of de novo glycosylation by tunicamycin blocked the secretion of Z-type alpha 1-antitrypsin completely.     

Intracellular processing of apolipoprotein J precursor to the mature heterodimer.

Circulating apolipoprotein J (apoJ) is a 70 kDa glycoprotein comprised of disulfide-linked alpha and beta subunits derived from a single precursor. Post-translational modifications that occur prior to apoJ secretion were assessed, with specific focus on carbohydrate type, the timing of proteolytic cleavage, and the importance of glycosylation on the cleavage and secretion processes. ApoJ was initially resolved as a single chain, intracellular precursor of 58 kDa which contained N-linked oligosaccharide but no O-linked oligosaccharide. The precursor was converted to an intracellular 70 kDa glycoprotein, which became the major intracellular form of apoJ prior to secretion. Maturation of the 58 kDa precursor involved conversion of high-mannose carbohydrate to complex-type carbohydrate containing sialic acid, as well as intracellular cleavage to yield alpha and beta subunits. This cleavage event occurred at a late stage of carbohydrate modification, most likely in the trans-Golgi or a post-Golgi compartment. The maturation and secretion of apoJ occurred rapidly, with a half-time of 30-35 min. Tunicamycin treatment of cells resulted in an unglycosylated doublet comprised of one single chain and one cleaved form of apoJ. The unglycosylated apoJ species were secreted rapidly with a half-time of 20 min. Both cleavage and secretion were independent of glycosylation.     

Production and secretion of interleukin 1 receptor antagonist in monocytes and keratinocytes

IL-1 receptor antagonist (IL-1ra) is a newly described member of the IL-1 family, isolated from supernatants of Ig stimulated monocytes, that binds competitively to IL-1 receptors without stimulating target cells (1–3). Also epithelial cells produce IL-1ra in a form which lacks a secretory signal sequence (4).Here we have compared the biosynthesis and secretion of IL-1ra in monocytes and keratinocytes. Our data show that monocytes produce two molecular forms of IL-1ra, of 18 Kd and 23 Kd respectively, which differ in the degree of glycosylation. Both forms are secreted via the “classical” endoplasmic reticulum (ER)-Golgi secretory pathway. By contrast keratinocytes produce IL-1ra in a molecular form of 20 Kd, which is not N-glycosylated: 20 Kd IL-1ra is detectable in supernatants of keratinocytes, although in small amounts. The presence of IL-1ra in keratinocytes cultures fluids is not inhibited by Brefeldin A (BFA), suggesting a possible secretion through the leaderless secretory pathway.     

Identification of an interleukin 17F/17A heterodimer in activated human CD4+ T cells

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.     

A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence

Interleukin 1 (IL-1) is a major soluble mediator of inflammation. Two human IL-1 genes, alpha and beta, have been isolated, which encode polypeptides with only 20-30% amino acid sequence homology. Unlike most secreted proteins, the two cytokines do not have a signal sequence, an unexpected finding in view of their biological role. Here we show that IL-1 beta is actively secreted by activated human monocytes via a pathway of secretion different from the classical endoplasmic reticulum--Golgi route. Drugs which block the intracellular transport of IL-6, of tumour necrosis factor alpha and of other secretory proteins do not inhibit secretion of IL-1 beta. Secretion of IL-1 beta is blocked by methylamine, low temperature or serum free medium, and is increased by raising the culture temperature to 42 degrees C or by the presence of calcium ionophores, brefeldin A, monensin, dinitrophenol or carbonyl cyanide chlorophenylhydrazone. IL-1 beta is contained in part within intracellular vesicles which protect it from protease digestion. In U937 cells large amounts of IL-1 beta are made but none is secreted. In these cells IL-1 beta is not found in the vesicular fraction, and all the protein is accessible to protease digestion. This suggests that intracellular vesicles that contain IL-1 beta are part of the protein secretory pathway. We conclude that IL-1 beta is released by activated monocytes via a novel mechanism of secretion which may involve translocation of intracellular membranes and is increased by stress conditions.     

A second subunit of CD8 is expressed in human T cells.

The CD8 glycoprotein plays important functions in T cell development and in T cell activation. In rodents, CD8 is a heterodimer, consisting of an alpha-chain (Lyt2) and a beta-chain (Lyt3). In humans, only the alpha-chain has been detected, and it has been thought that CD8 consists of homodimers of this protein. We have isolated functional cDNA clones encoding human CD8 beta, and show that the CD8 beta protein is expressed on the surface of CD8+ human T cells. cDNA clones encoding multiple forms of the human CD8 beta-chain have been isolated and characterized. These structural variants, which are likely to arise by alternative splicing, differ in the sequences encoding the cytoplasmic domain, which can consist of 19, 30, or 52 amino acids. One of the cDNAs lacks nucleotide sequences corresponding to a hydrophobic transmembrane domain, and may encode a secreted CD8 beta protein. The protein product of the human CD8 beta gene can be detected by a recently described anti-CD8 monoclonal antibody, 597. Expression of the epitope recognized by this antibody requires co-expression of the CD8 alpha and CD8 beta gene products. About 90% of human CD8 alpha positive thymocytes and peripheral blood lymphocytes express CD8 beta at the cell surface. Expression of the CD8 beta chain is thus conserved between human and rodents, and the variant CD8 beta polypeptides may have distinct roles in T cell function and development.     

Induction of interleukin-12 (IL-12) by recombinant glycoprotein gp120 of human immunodeficiency virus type 1 in human monocytes/macrophages: requirement of gamma interferon for IL-12 secretion.

We studied the effects of the gp120 glycoprotein of human immunodeficiency virus type 1 on the expression of interleukin-12 (IL-12) in human monocytes and in monocyte-derived macrophages. Induction of the mRNA for both the p35 and p40 subunits of IL-12 was observed in both cell types after gp120 treatment. We then evaluated cytokine secretion by using an enzyme-linked immunosorbent assay which recognizes only the IL-12 heterodimer. No IL-12 was detected in monocytes/macrophages treated with gp120 alone. A consistent IL-12 secretion was found in macrophages primed with gamma interferon (IFN-gamma) and subsequently treated with gp120. Low levels of IL-12 were occasionally observed in IFN-gamma-primed monocytes stimulated with gp120. The greater response of macrophages than of monocytes to the priming effect of IFN-gamma was consistent with the finding that IFN-gamma induced a much stronger antiviral state to vesicular stomatitis virus in macrophages than in monocytes. These data indicate that gp120 is an inducer of IL-12 expression in monocytes/macrophages and that IFN-gamma is an essential cofactor for IL-12 secretion, especially in differentiated macrophages.     

Differential glycosylation of two glycoproteins synthesized by murine B cells in response to IL-4 plus IL-5

We sought to determine whether selected cytokines, known to stimulate profoundly B-cell activation and differentiation, also have as yet unrecognized effects upon the glycosylation of secreted Ig and/or membrane-associated proteins. The glycosylation of both secreted IgM and membrane-bound MHC Class-I synthesized by CH12LX cells was detected by enzyme-lectin conjugates in immunoabsorption assays. Stimulation of B cells with IL-4 plus IL-5 significantly decreases the terminal glycosylation of secreted IgM, whereas LPS has a minor effect, despite the fact that both stimuli are equipotent for IgM secretion. Neither LPS nor IL-4 plus IL-5 affect MHC Class-I expression. However, IL-4 plus IL-5 substantially increases the terminal glycosylation of MHC Class-I produced from both mIgM(+)and mIgA(+)CH12LX cells. LPS has no or a modest effect on the terminal glycosylation of MHC Class-I produced from CH12LX cells. These results suggest that Th(2)-derived cytokines differentially influence the glycosylation of secreted and membrane-associated glycoproteins of B cells. In turn, this might elucidate the basis of aberrant glycosylation reported in conditions such as IgA nephropathy, cancer and rheumatoid arthritis.     

Regulation of neutrophil interleukin 8 gene expression and protein secretion by LPS,TNF-α, and IL-1β

Neutrophils are possibly involved in the pathogenesis of various lung diseases through the release of numerous mediators. In the present study, we studied the regulation of IL-8 gene induction and protein secretion in human blood neutrophils. Northern blot analysis revealed that LPS increased IL-8 mRNA levels in neutrophils, with a maximal fivefold increase by 2 h. IL-8 mRNA levels returned to baseline value within 12 h. In contrast, LPS-stimulated monocytes demonstrated a sustained increase of IL-8 mRNA levels for more than 24 h. TNF-α, IL-1β, and phorbol myristate acetate also increased IL-8 mRNA levels in neutrophils. Immunohistochemical analysis confirmed that IL-8 was localized within stimulated neutrophils. IL-8 secretion by neutrophils and monocytes was quantified using a specific ELISA for IL-8. Resting neutrophils secreted minimal IL-8 activity. However when cells were stimualted with LPS, TNF-α, or IL-1bT, neutrophils secreted IL-8. IL-8 secretion was most marked during the first 2 h after stimulation and decreased thereafter. In contrast, monocytes maintained a high rate of IL-8 secretion over 12 h. Although a single monocyte secreted 70-fold more IL-8 than did a single neutrophil after 4 h of incubation, the high abundance of neutrophils in peripheral blood made the neutrophil-secreted IL-8 more significant. During the first 2 h, neutrophils secreted ～40% of the IL-8 released by monocytes in the same volume of blood. This ratio decreased to 9% after 12 h. Neutrophil-secreted IL-8 may play an autocrine or paracrine role during the initial stage of inflammation. © 1993 Wiley-Liss, Inc.     

Distinct aggregation of beta- and gamma-chains of the high-affinity IgE receptor on cross-linking.

The high-affinity IgE receptor (FcepsilonRI) on mast cells and basophils consists of a ligand-binding alpha-chain and two kinds of signaling chains, a beta-chain and disulfide-linked homodimeric gamma-chains. Crosslinking by multivalent antigen results in the aggregation of the bound IgE/alpha-chain complexes at the cell surface, triggering cell activation, and subsequent internalization through coated pits. However, the precise topographical alterations of the signaling beta- and gamma-chains during stimulation remain unclarified despite their importance in ligand binding/signaling coupling. Here we describe the dynamics of FcepsilonRI subunit distribution in rat basophilic leukemia cells during stimulation as revealed by immunofluorescence and immunogold electron microscopy. Immunolocalization of beta- and gamma-chains was homogeneously distributed on the cell surfaces before stimulation, while crosslinking with multivalent antigen, which elicited optimal degranulation, caused a distinct aggregation of these signaling chains on the cell membrane. Moreover, only gamma- but not beta-chains were aggregated during the stimulation that evoked suboptimal secretion. These findings suggest that high-affinity IgE receptor beta- and gamma-chains do not co-aggregate but for the most part form homogenous aggregates of beta-chains or gamma-chains after crosslinking.     

Production and secretion of interleukin 1 receptor antagonist in monocytes and keratinocytes

IL-1 receptor antagonist (IL-1ra) is a newly described member of the IL-1 family, isolated from supernatants of Ig stimulated monocytes, that binds competitively to IL-1 receptors without stimulating target cells (1–3). Also epithelial cells produce IL-1ra in a form which lacks a secretory signal sequence (4).Here we have compared the biosynthesis and secretion of IL-1ra in monocytes and keratinocytes. Our data show that monocytes produce two molecular forms of IL-1ra, of 18 Kd and 23 Kd respectively, which differ in the degree of glycosylation. Both forms are secreted via the classical endoplasmic reticulum (ER)-Golgi secretory pathway. By contrast keratinocytes produce IL-1ra in a molecular form of 20 Kd, which is not N-glycosylated: 20 Kd IL-1ra is detectable in supernatants of keratinocytes, although in small amounts. The presence of IL-1ra in keratinocytes cultures fluids is not inhibited by Brefeldin A (BFA), suggesting a possible secretion through the leaderless secretory pathway.     

The kinetics of interleukin 1 secretion from activated monocytes. Differences between interleukin 1 alpha and interleukin 1 beta

We have performed pulse-chase experiments to investigate the secretion and processing of interleukin 1 (IL-1) by human peripheral blood monocytes. Polyclonal antisera generated against either recombinant IL-1 alpha (p15) or IL-1 beta (p17) could distinguish the two isoelectric forms in lysates and supernatants of lipopolysaccharide-activated monocytes. In agreement with previous results, no processed IL-1 (alpha or beta) is detected in cell lysates. Both the 31-kDa precursor and 17-kDa mature forms of IL-1 were present, however, in the culture media indicating that processing is not required for secretion. The relative amounts of the secreted 31- and 17-kDa forms of IL-1 remain constant with time throughout each experiment; in addition, 31-kDa IL-1 added to monocyte cultures is not processed to the mature 17-kDa form. Precursor IL-1 beta is however, processed to 17 kDa by monocyte extracts. Therefore, the maturation and secretion of IL-1 are intimately coordinated processes. The kinetics of IL-1 secretion are unique in comparison with other secreted proteins; release of both IL-1 alpha and IL-1 beta is delayed following synthesis, and large pools of precursor IL-1 accumulate intracellularly. The intracellular half-lives of IL-1 alpha and IL-1 beta are 15 and 2.5 h, respectively. This discrepancy in half-lives is a reflection of the different kinetics with which IL-1 alpha and IL-1 beta are secreted. IL-1 beta is released continuously beginning 2 h after synthesis, whereas the secretion of IL-1 alpha is delayed for an additional 10 h. The distinct kinetics of secretion demonstrated for IL-1 alpha and IL-1 beta suggest that the release of each pI species of IL-1 is controlled by a selective mechanism(s).     

Location of the interchain disulfide bonds of the fourth component of human complement (C4): evidence based on the liberation of fragments secondary to thiol-disulfide interchange reactions

Treatment of human C4 with chemical denaturants and heat produces rapid, autolytic peptide bond cleavage of the alpha-chain. These alpha-chain fragments are linked to the parent C4 molecule through disulfide bonds. On more prolonged heating, however, there is liberation of several peptides, including the beta-chain, the gamma-chain, and a C-terminal alpha-chain fragment. This reaction is inhibited by iodoacetamide. By using a fluorescent thiol reagent and 14C-iodoacetamide, the thiol group present on each peptide was analyzed. The results suggest that the thiol residue exposed by cleavage of the thioester bond induces thiol-disulfide interchange reactions to liberate the peptides. Based on the identification of fragments liberated, the kinetics of their appearance, their sulfhydryl content, and the reported primary structure of human C4, a model of the interchain disulfide bonds is proposed in which the amino terminal portion of the alpha-chain is disulfide-linked to both the beta- and gamma-chains, whereas the carboxyl terminal portion of the alpha-chain is disulfide-linked to only the gamma-chain.     

Reconstitution of a functional IL-2 receptor by the beta-chain cDNA. A newly acquired receptor transduces negative signal

The high-affinity IL-2R results from the noncovalent association between at least two subunits; alpha (p55) and beta (p70), both of which are capable of binding IL-2 with a low and intermediate affinity, respectively. Although the alpha-chain itself has been shown to be nonfunctional, suggestions have been made that the beta-chain mediates an IL-2 signal. To directly study the role of the beta-chain in the signal transduction, we transfected with the cDNA encoding the IL-2R beta-chain a human T lymphotropic virus-I-transformed T cell line, MT-1 originally expressing low-affinity alpha-chain alone, and established a stable transformant (designated MT-beta 7) which expressed both alpha- and beta-chains simultaneously. We showed 1) MT-beta 7 manifested the high-affinity IL-2 binding, which was completely disrupted by the anti-beta chain mAb (Mik-beta 1), 2) the 125I-IL-2 crosslinking patterns of MT-beta 7 were indistinguishable from those of cells expressing the native high-affinity IL-2R, 3) MT-beta 7, but not parental MT-1, internalized the bound IL-2 and responded to IL-2 with a negative signal, i.e., inhibition of the de novo DNA synthesis. These results clearly demonstrate that the beta-chain not only participates in forming the high-affinity IL-2R with the alpha-chain but also is directly involved in the IL-2 signal transduction.     

Disruption of N-linked glycosylation of bovine luteinizing hormone beta-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer

The single site for N-linked glycosylation of the beta-subunit of bovine LH (LH beta) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH alpha/beta heterodimer. Pulsechase studies performed with stably transfected Chinese hamster ovary cells that expressed both alpha-subunit (fully glycosylated) and nonglycosylated LH beta revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LH beta were effectively blocked over a 22-h span. Free nonglycosylated LH beta, like free wild-type LH beta, was sequestered inside the cell; therefore, the intracellular retention of uncombined LH beta-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LH beta-subunit was available for combination with newly synthesized alpha-subunit, as verified by immunoprecipitation of radiolabeled alpha-subunit from cell lysates and culture medium with anti-LH beta-antiserum. This heterodimer displayed normal kinetics of secretion (t 1/2 = 2.4 h) as compared to fully glycosylated LH (t 1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release Pmax = 2.7 vs 2.5 micrograms/ml), indicating that N-linked glycosylation of the LH beta-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LH beta, but is not essential for either its assembly with alpha-subunit or the transport and secretion of biologically active heterodimer.