Alvin S. Stern

Putative enkephalin precursors in bovine adrenal medulla.

Abstract Extracts from bovine adrenal medulla and adrenal medullary chromaffin granules were found to contain three proteins, 20,000, 10,000 and 5,000 approximate molecular weights which yield tryptic peptides with opioid activity. The opioid activity of these peptides was demonstrated with a radioreceptor assay and two radioimmunoassays. The three proteins yield the same active peptides all of which are chromatographically distinct from the tryptic opioid nonapeptide β-LPH 61–69, generated by trypsin digestion of pituitary endorphins and their precursors. Furthermore, these endorphins and their precursors do not appear to be present in the adrenal medulla. These findings further support the hypothesis that the enkephalin biosynthetic pathway is distinct from that leading to β-endorphin.

Interleukin-12 Antagonist Activity of Mouse Interleukin-12 p40 Homodimer in Vitro and in Vivo

Mo(p40)2 is a potent IL-12 antagonist that interacts strongly with the beta 1 subunit of the IL-12R to block binding of moIL-12 to the high-affinity mouse IL-12R. Mo(p40)2, alone or in synergy with the 2B5 mAb specific for the moIL-12 heterodimer, blocked IL-12-induced responses in vitro, Mo(p40)2 was thus used alone or with 2B5 mAb to examine the role of IL-12 in vivo, Mo(p40)2 caused a dose-dependent inhibition of both the rise in serum IFN-gamma levels in mice injected with endotoxin and the Th1-like response to immunization with KLH. Treatment with mo(p40)2 plus 2B5 anti-moIL-12 mAb also suppressed DTH responses to methylated bovine serum albumin but not specific allogeneic CTL responses in vivo. In each of these models, responses seen in mice treated with mo(p40)2 +/- 2B5 anti-moIL-12 mAb were similar to those observed in IL-12 knockout mice. Thus, mo(p40)2 can act as a potent IL-12 antagonist in vivo, as well as in vitro, and is currently being used to investigate the role of IL-12 in the pathogenesis of some Th1-associated autoimmune disorders in mice.

Molecular characterization of interleukin 12

Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor, is a cytokine secreted by a human B lymphoblastoid (NC-37) cell line when induced in culture with phorbol ester and calcium ionophore. This factor has been purified to homogeneity and shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells. In addition, purified IL-12 stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. The protein is a heterodimer composed of a 40- and a 35-kDa subunit. Amino acid sequence analysis confirmed predicted sequences from the cloned cDNAs of each subunit. Chemical and enzymatic deglycosylation of the heterodimer demonstrated that the 40- and 35-kDa subunits contain 10 and 20% carbohydrate, respectively. Structural analysis of IL-12 using site-specific chemical modification revealed that intact disulfide bonds are essential for bioactivity. The 40-kDa subunit of IL-12 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by immunoblotting as being present in NC-37 cell supernatant solutions in relatively large amounts uncomplexed to the 35-kDa subunit. Previously it had been shown that the 40-kDa subunit alone does not cause the proliferation of activated human T lymphocytes or enhance the cytolytic activity of human natural killer cells. However, results obtained by site-specific chemical modification suggesting that a tryptophan residue is at or near the active site of IL-12 may imply a direct role of the subunit in interacting with the IL-12 receptor. These data may support the recent proposal (D.P. Gearing and D. Cosman (1991) Cell 66, 9-10) that IL-12 consists of a complex of cytokine and soluble receptor.

INTERLEUKIN-12 AN INTEGRAL CYTOKINE IN THE IMMUNE RESPONSE

Interleukin 12 (IL-12) is a heterodimeric cytokine that is produced primarily by antigen-presenting cells and plays a primary role in the induction of cell-mediated immunity. This function is promoted by the IL-12 induced production of interferon-gamma (IFN-gamma) from both resting and activated NK and T cells, by the proliferative activity of IL-12 on activated NK and T cells, by enhancing the cytotoxic activity of NK cells, and by supporting cytotoxic T lymphocyte generation. IL-12 and IL-12-induced IFN-gamma promote the development of naive T cells into Th1 cells and the proliferation and IFN-gamma secretion by differentiated Th1 cells in response to antigen. IL-12 has been found to exhibit many of these activities in vivo, as well as in vitro, and thus IL-12 plays an important role in both innate resistance and antigen-specific adaptive immunity to intracellular bacterial, fungal, and protozoan pathogens. Due to its effects on T cells, recombinant IL-12 has been shown to have therapeutic activity in a variety of mouse tumor and infectious disease models and is being evaluated in clinical trials in human cancer patients. IL-12 also appears to play a role in the genesis of some forms of immunopathology, including endotoxin-induced shock and some autoimmune diseases associated with aberrant Th1 activity. Therefore, IL-12 antagonists may also have therapeutic potential in the treatment of auto immune disorders.

Opioid hexapeptides and heptapeptides in adrenal medulla and brain possible implications on the biosynthesis of enkephalins

Bovine adrenal chromaffin granules have been shown to contain [Met]enkephalin and [Leu]enkephalin and at least seven other small peptides that exhibit specific binding to opiate receptors. Six of these peptides have been characterized and their structures established as (O)-[Met]enkephalin, [Met]enkephalin-Arg6-Arg7, [Met]enkephalin-Lys6, [Met]enkephalin-Arg6, [Leu]enkephalin-Arg6, and [Met]enkephalin-Arg6-Phe7. Many of these hexa- and heptapeptides are also present in bovine and human brain. It is suggested that the presence of these peptides in a tissue is evidence of a common biosynthetic pathway of the enkephalins from a large precursor protein.

Structure of two adrenal polypeptides containing multiple enkephalin sequences

Abstract Two enkephalin-containing polypeptides of 4000 and 5000 daltons have been isolated from extracts of bovine adrenal medulla. Each polypeptide was purified to homogeneity and subjected to sequence analysis. The entire primary structure of the 4000-dalton polypeptide was established by a combination of automated Edman degradation and chemical analysis of its tryptic peptides. The polypeptide contains two copies of the [Met]-enkephalin sequence, one at the amino terminus and the other at the carboxyl terminus. Chemical analysis of the tryptic peptides and automated Edman degradation of the 5000-dalton polypeptide indicated the presence of a [Leu]enkephalin sequence at the carboxyl terminus and an internal [Met]enkephalin sequence. Both of the above enkephalin-containing polypeptides appear to be intermediates in the biosynthesis of the enkephalins.

Proenkephalin: A general pathway for enkephalin biosynthesis in animal tissues

Guinea pig adrenal, brain, and myenteric plexus have been shown to contain many polypeptides that yield free enkephalins on digestion with trypsin and carboxypeptidase B. The enkephalin-containing polypeptides (ECPs) range from 500 to >20,000 daltons and show similarities in their chromatographic behavior to the ECPs present in the chromaffin granules of the bovine adrenal medulla. Furthermore, the heptapeptide [Met]enkephalin-Arg6-Phe7, that is now known to represent the carboxyl terminal sequence of the proenkephalin found in bovine adrenal medulla (Gubler et al. (1982) Nature (London), in press), was identified in all three guinea pig tissues. It appears that processing of a proenkephalin similar to the one in adrenal medulla represents a general pathway for enkephalin biosynthesis in animal tissues.

Characterization of mouse interleukin-12 p40 homodimer binding to the interleukin-12 receptor subunits

Interleukin‐12 (IL‐12) is a heterodimeric cytokine composed of two disulfide‐bonded subunits, p35 and p40, which has important regulatory effects on T cells and natural killer (NK) cells. In contrast to heterodimeric IL‐12, a homodimer of the p40 subunit, designated (p40)2, has been shown to be a potent IL‐12 antagonist. To study the interaction between (p40)2 and the known IL‐12 receptor (IL‐12R) subunits, IL‐12Rβ1 and IL‐12Rβ2, we directly measured the binding activity of mouse (p40)2 to ConA‐activated lymphoblasts and purified B cells from splenocytes of C57BL/6J mice. These results demonstrated the presence of both high (Kd about 5 pM) and low affinity (Kd about 15 nM) binding sites for mouse 125I‐labeled (p40)2. To elucidate which of the IL‐12R subunits binds mouse (p40)2, binding studies of mouse 125I‐labeled (p40)2 to Ba/F3 cells expressing recombinant mouse IL‐12Rβ1 and/or mouse IL‐12Rβ2 were carried out. Mouse IL‐12Rβ1 bound mouse 125I‐labeled (p40)2 with high and low affinities, comparable to that observed on Con A blasts and B cells. In contrast, mouse IL‐12Rβ2 bound mouse 125I‐labeled (p40)2 very poorly. These data demonstrate that similar to IL‐12, mouse (p40)2 binds with both high and low affinity to Con A blasts and B cells, and that IL‐12Rβ1 is responsible for mediating the specific binding of mouse (p40)2.

Evidence for Multiple Sites of Interaction between IL‐12 and Its Receptor

We have previously described the identification of a protein, now designated IL-12R beta 1, that binds 125I-huIL-12 with a Kd of about 10 nM, corresponding to the low affinity 125I-huIL-12 binding sites seen on PHA-activated human lymphoblasts. Using expression cloning techniques, we have recently identified an additional IL-12-binding protein subunit, IL-12R beta 2, which binds 125I-huIL-12 with a Kd of about 5 nM when expressed alone in COS-7 cells. Coexpression of IL-12R beta 1 and IL-12R beta 2 in COS-7 cells results in formation of two classes of 125 I-huIL-12-binding sites with Kds of about 50 pM and 5 nM. Mouse IL-12 p40 subunit homodimer (mo(p40)2) blocked 125I-huIL-12 binding to human IL-12R beta 1, but did not inhibit binding to human IL-12R beta 2. In contrast, anti-huIL-12 monoclonal antibody 20C2, which does not block 125I-huIL-12 binding to human IL-12R beta 1, completely blocked binding to human IL-12R beta 2. These results demonstrate that two classes of IL-12 inhibitors, one that primarily blocks IL-12/IL-12R beta 1 interaction (e.g., mo(p40)2), and one that primarily blocks IL-12/IL-12R beta 2 interaction (e.g., 20C2), can be identified.

Allosteric antibody inhibition of human hepsin protease

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation.