Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion

Mesothelin is a glycosylphosphatidylinositol-linked cell surface molecule expressed in the mesothelial lining of the body cavities and in many tumor cells. Based on the finding that a soluble form of mesothelin specifically binds to ovarian carcinoma cell line OVCAR-3, we isolated cDNAs encoding a mesothelin-binding protein by expression cloning. The polypeptides encoded by the two cloned cDNA fragments matched to portions of CA125, an ovarian cancer antigen and a giant mucin-like glycoprotein present at the surface of tumor cells. By flow cytometric analysis and immunoprecipitation, we demonstrate that CA125 binds to mesothelin in a specific manner. Binding of CA125 to membrane-bound mesothelin mediates heterotypic cell adhesion as anti-mesothelin antibody blocks binding of OVCAR-3 cells expressing CA125 to an endothelial-like cell line expressing mesothelin. Finally, we show that CA125 and mesothelin are co-expressed in advanced grade ovarian adenocarcinoma. Taken together, our data indicate that mesothelin is a novel CA125-binding protein and that CA125 might contribute to the metastasis of ovarian cancer to the peritoneum by initiating cell attachment to the mesothelial epithelium via binding to mesothelin.     

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.     

Engineered Fn3 protein has targeted therapeutic effect on mesothelin-expressing cancer cells and increases tumor cell sensitivity to chemotherapy

Mesothelin is a protein expressed at high levels on the cell surface in a variety of cancers, with limited expression in healthy tissues. The presence of mesothelin on tumor tissue correlates with increased invasion and metastasis, and resistance to traditional chemotherapies, through mechanisms that remain poorly understood. Molecules that specifically recognize mesothelin and interrupt its contribution to tumor progression have significant potential for targeted therapy and targeted drug delivery applications. A number of mesothelin-targeting therapies are in preclinical and clinical development, although none are currently approved for routine clinical use. In this work, we report the development of a mesothelin-targeting protein based on the fibronectin type-III non-antibody protein scaffold, which offers opportunities for applications where antibodies have limitations. We engineered protein variants that bind mesothelin with high affinity and selectively initiate apoptosis in tumor cells expressing mesothelin. Interestingly, apoptosis does not occur through a caspase-mediated pathway and does not require downregulation of cell-surface mesothelin, suggesting a currently unknown pathway through which mesothelin contributes to cancer progression. Importantly, simultaneous treatment with mesothelin-binding protein and chemotherapeutic mitomycin C had a greater cytotoxic effect on mesothelin-positive cells compared to either molecule alone, underscoring the potential for combination therapy including biologics targeting mesothelin.     

Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples

Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified “HV4” and “LV4” in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.     

Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins

A low-affinity adenosine binding protein has recently been distinguished from the adenosine A2 receptor and purified from human placental membranes. Soluble human placental extracts contain an adenosine binding activity that has properties similar to those of the membrane low-affinity adenosine binding protein. The binding protein was purified from soluble human placental extracts 134-fold to 89% purity with a Bmax of 2.5 nmol/mg. It comprises 0.7-0.9% of the soluble protein. The major purified soluble protein has a subunit molecular mass of 98 kDa and a Stokes radius identical with that of the membrane-bound adenosine binding protein. Competition analysis of the soluble protein revealed similar affinities and an identical potency order for displacement of 5'-(N-ethylcarbamoyl)[2,8-3H]adenosine ([3H]NECA) as follows: NECA greater than 2-chloroadenosine greater than adenosine greater than (R)-N6-(2-phenylisopropyl)adenosine. The soluble binding protein was more acidic than the membrane binding protein as revealed by a comparison of the elution properties during ion exchange chromatography. A second form of soluble adenosine binding activity comprised 17% of the major form and had a charge similar to that of the membrane binding protein, a smaller Stokes radius, and a subunit molecular mass of 74 kDa. Carbohydrate composition analysis revealed that the major soluble form has 4.3% carbohydrate by weight as compared to the membrane-associated form, which has 5.5% carbohydrate by weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid identification of recombinant Fabs that bind to membrane proteins

Crystallographic studies of membrane proteins have been steadily increasing despite their unique physical properties that hinder crystal formation. Co-crystallization with antibody fragments has emerged as a promising solution to obtain diffraction quality crystals. Antibody binding to the target membrane protein can yield a homogenous population of the protein. Interantibody interactions can also provide additional crystal contacts, which are minimized in membrane proteins due to micelle formation around the transmembrane segments. Rapid identification of antibody fragments that can recognize native protein structure makes phage display a valuable method for crystallographic studies of membrane proteins. Methods that speed the reliable characterization of phage display selected antibody fragments are needed to make the technology more generally applicable. In this report, a phage display biopanning procedure is described to identify Fragments antigen binding (Fabs) for membrane proteins. It is also demonstrated that Fabs can be rapidly grouped based on relative affinities using enzyme linked immunosorbent assay (ELISA) and unpurified Fabs. This procedure greatly speeds the prioritization of candidate binders to membrane proteins and will aid in subsequent structure determinations.     

Structural comparisons between the soluble and the GPI-anchored forms of the Paramecium temperature-specific 156G surface antigen.

Biosynthetic labelling experiments performed on P primaurelia strain 156, expressing the temperature-specific G surface antigen, 156G SAg, demonstrated that the purified 156G SAg contained the components characteristic of a GPI-anchor. [3H]ethanolamine, [3H]myo-inositol, [32P]phosphoric acid and [3H]myristic acid could all be incorporated into the surface antigen. Myristic acid labelling was lost after treatment in vitro with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). After complete digestion by pronase, a fragment containing the intact GPI-anchor of 156G surface antigen was isolated. This fragment was shown to be hydrophobic and glycosylated and to possess an epitope found specifically in the GPI component of GPI-anchored proteins. The role of the GPI-tail in anchoring the 156G surface antigen into the membrane was assessed by determining that purified 156G molecules with the GPI-anchor could be incorporated into lipid vesicles and red cell ghosts whereas the 156G molecules lacking the GPI-anchor, as result of treatment with B thuringiensis PI-PLC, could not. It has also been shown that the membrane-bound form and the soluble form, obtained after cleavage of the 156G SAg lipid moiety either by an endogenous PI-PLC or by a bacterial PI-PLC, displayed identical circular dichroic spectra.     

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.     

Growth factor induction of Cripto-1 shedding by glycosylphosphatidylinositol-phospholipase D and enhancement of endothelial cell migration

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.     

Myristoylation, phosphorylation, and subcellular distribution of the 80-kDa protein kinase C substrate in BC3H1 myocytes

Numerous reports have described a phosphoprotein with an apparent molecular mass of 68-87 kDa, often referred to as the 80K protein, which serves as a major specific substrate for protein kinase C in a wide variety of cell types. This protein has been shown to be myristoylated in macrophages, apparently in a stimulus-dependent manner. In the present study, we have defined the kinetics for myristoylation of the 80K protein in BC3H1 myocytes and have examined the subcellular distribution of the [3H]myristate and 32P-labeled forms of the protein before and after activation of protein kinase C by phorbol dibutyrate (PDBu). The 80K protein was identified in BC3H1 myocytes by apparent molecular mass of 68 kDa (consistent with the previously reported size of the murine homologue), isoelectric point of 4.6-4.8, PDBu-inducible phosphorylation, peptide mapping, and labeling with [3H]myristate. Incorporation of [3H]myristate by this protein occurred through an amide linkage and was abolished completely by cycloheximide. Pulse labeling of quiescent cells with [3H]myristate revealed no alteration in myristoylation of the 80K protein in either the crude membrane or soluble fractions after PDBu-induced phosphorylation. The subcellular distribution of this protein (approximately 80% membrane, approximately 20% cytosol) also was the same in control and PDBu-stimulated cells. Phosphorylation of both the membrane-bound and soluble forms was increased approximately 6-fold upon stimulation of cultures with PDBu; the soluble form was phosphorylated to a 4-fold higher stoichiometry than its membrane-bound counterpart. Together, these data demonstrate that the 80K protein is myristoylated cotranslationally in BC3H1 cells and that protein kinase C-dependent phosphorylation of the 80K protein does not alter its subcellular distribution or degree of myristoylation. The fact that 20% of total myristoylated 80K protein resides in the cytosol also indicates that myristoylation alone is not sufficient to target this protein to the plasma membrane.     

Vanadate inhibits degradation of short-lived, but not of long-lived, proteins in L-132 human cells.

We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms.     

Posttranslational Processing of Carboxypeptidase E, a Neuropeptide-Processing Enzyme, in AtT-20 Cells and Bovine Pituitary Secretory Granules

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, “proCPE.” To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [³⁵S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCI₂. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.     

Dissociation of G-protein alpha from rhabdomeric membranes decreases its interaction with rhodopsin and increases its degradation by calpain

Photoactivation of invertebrate rhodopsin activates a GTP-binding protein, Gq, which in turn activates a phospholipase C (PLC) enzyme. Gqalpha is a membrane-associated protein that is progressively released from the membrane by washing with buffers containing increasing concentrations of beta-mercaptoethanol (beta-ME). Isolated, soluble Gqalpha showed a decreased ability to be activated by rhodopsin but was more active in stimulating PLC when compared with the membrane-associated form of Gqalpha. The calcium-activated protease, calpain, selectively cleaved the soluble but not the membrane-bound form of Gqalpha. Calpain cleaved a small peptide from the amino-terminus of Gqalpha reducing the ability of the G-protein to bind GTP. The uncoupling of Gqalpha from rhodopsin and subsequent calcium-dependent proteolysis to further inactivate the G-protein may therefore be a regulatory mechanism of light adaptation in rhabdomeric photoreceptors.     

The membrane-anchor of Paramecium temperature-specific surface antigens is a glycosylinositol phospholipid

The temperature-specific G surface antigen of Paramecium primaurelia strain 156 was biosynthetically labeled by [3H]myristic acid in its membrane-bound form, but not in its soluble form. It could be cleaved by a phosphatidylinositol-specific phospholipase C from Trypanosoma brucei or from Bacillus cereus which released its soluble form with the unmasking of a particular glycosidic immunodeterminant called the crossreacting determinant. The Paramecium enzyme, capable of converting its membrane-bound form into the soluble one, was inhibited by a sulphydril reagent in the same way as the trypanosomal lipase. From this evidence we propose that the Paramecium temperature-specific surface antigens are anchored in the plasma membrane via a glycophospholipid, and that an endogenous phospholipase C may be involved in the antigenic variation process.     

Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol.

Alkaline phosphatase is anchored to the membrane via glycosylphosphatidylinositol (GPI). Mannose residues of the GPI glycan are suggested to be derived from dolichol-P-mannose. In the present study we examined the effect of 2-fluoro-2-deoxy-D-glucose (F-Glc), an inhibitor of dolichol-P-mannose synthesis, on the biosynthesis and processing of alkaline phosphatase in JEG-3 cells. In control cells, a proform precursor (64.5 kDa) with a hydrophobic peptide domain at the COOH terminus was immediately processed into an intermediate form (63 kDa) by proteolytic removal of the COOH-terminal extension and replacement with the GPI anchor, and then to a mature form (66 kDa) by terminal glycosylation of its N-linked oligosaccharides. In contrast, when cells were treated with F-Glc (1 mM), the protein was synthesized as a proform of 61 kDa. The reduction in its molecular mass was mostly due to the inhibition in maturation of N-linked oligosaccharides by F-Glc. The 61-kDa proform identified by antibodies to the COOH-terminal peptide was detectable even at 3 h after the synthesis, and was gradually processed to doublet forms of 58-59 kDa which were finally secreted into the medium. None of these forms were labeled with [3H]ethanolamine and [3H]stearic acid, components of the GPI anchor, and expressed on the cell surface as a membrane-bound form. Taken together, these results suggest that the inhibition of the GPI synthesis causes a prolonged accumulation of the proform, which is then gradually processed into secretory forms by proteolytic removal of the COOH-terminal hydrophobic peptide.     

Cholesterol effects on BAX pore activation

The importance of BCL-2 family proteins in the control of cell death has been clearly established. One of the key members of this family, BAX, has soluble, membrane-bound, and membrane-integrated forms that are central to the regulation of apoptosis. Using purified monomeric human BAX, defined liposomes, and isolated human mitochondria, we have characterized the soluble to membrane transition and pore formation by this protein. For the purified protein, activation, but not oligomerization, is required for membrane binding. The transition to the membrane environment includes a binding step that is reversible and distinct from the membrane integration step. Oligomerization and pore activation occur after the membrane integration. In cells, BAX targets several intracellular membranes but notably does not target the plasma membrane while initiating apoptosis. When cholesterol was added to either the liposome bilayer or mitochondrial membranes, we observed increased binding but markedly reduced integration of BAX into both membranes. This cholesterol inhibition of membrane integration accounts for the reduction of BAX pore activation in liposomes and mitochondrial membranes. Our results indicate that the presence of cholesterol in membranes inhibits the pore-forming activity of BAX by reducing the ability of BAX to transition from a membrane-associated protein to a membrane-integral protein.     

MESOMARK kit detects C-ERC/mesothelin, but not SMRP with C-terminus

ERC/mesothelin is expressed on the normal mesothelium and some cancers such as mesothelioma or ovarian carcinoma. A splicing isoform of ERC/mesothelin (known as SMRP), which has an 82-bp insertion and codes for a C-terminus with a hydrophilic, presumably soluble, tail instead of a GPI-anchoring signal, has been reported as a useful marker for the diagnosis of mesothelioma. However, the existence of SMRP has not yet been demonstrated in the serum of mesothelioma patients. To elucidate the existence of SMRP, we have established a new enzyme-linked immunosorbent assay (ELISA) system for SMRP. The ELISA study revealed that N- and C-ERC/mesothelin were detected in sera from mesothelioma patients, but not SMRP, even in these samples. This result showed that the SMRP detected with MESOMARK kit should be lack of soluble C-terminus and indistinguishable from C-ERC/mesothelin. Further study might be necessary to demonstrate the relationship between SMRP and mesothelin.     

Solubilisation of methane monooxygenase from Methylococcus capsulatus (Bath)

The membrane-bound (particulate) form of methane monooxygenase from Methylococcus capsulatus (Bath) has been solubilised using the non-ionic detergent dodecyl-beta-D-maltoside. A wide variety of detergents were tested and found to solubilise membrane proteins but did not yield methane monooxygenase in a form that could be subsequently activated. After solubilisation with dodecyl-beta-D-maltoside, enzyme activity was recovered using either egg or soya-bean lipids. Attempts to further purify the solubilized methane monooxygenaser protein into its component polypeptides were unsuccessful and resulted in complete loss of enzyme activity. The major polypeptides present in the solubilised enzyme had molecular masses of 49 kDa, 23 kDa and 22 kDa which were similar to those seen in crude extracts [Prior, S. D. & Dalton H. (1985) J. Gen. Microbiol. 131, 155-163]. Studies on substrate and inhibitor specificities indicated that the membrane-associated and solubilised forms of methane monooxygenase were quite similar to each other but differed substantially from the well-characterised soluble methane monooxygenase found in cells grown in a low copper regime and synthesised independently of the particulate methane monooxygenase.     

Nature of the anchors of membrane-bound aminopeptidase,amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells

Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.     

Removal of Qa-2 antigen alters the Ped gene phenotype of preimplantation mouse embryos.

Embryo survival is influenced by both genetic and environmental factors. Previous research in our laboratory has identified one gene associated with embryonic survival, the Ped gene, a gene that is linked to the major histocompatibility complex (MHC) of the mouse. The Ped gene has been shown to influence the rate of preimplantation embryonic cleavage division, as well as litter size, birth weight, and weaning weight. Genetic mapping of the Ped gene has located it in the Q region of the MHC and has suggested that possible Q region genes encoding the Ped gene are Q3, Q5, Q6, Q7, Q8, and/or Q9. Whereas the protein products of the Q3 and Q5 genes are unknown, the protein product of the very similar Q6, Q7, Q8, and Q9 genes is the Qa-2 antigen. Two forms of membrane-bound Qa-2 antigen are known: glycosylphosphatidylinositol (GPI)-linked and transmembrane bound. Only the GPI-linked form is sensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC). The first purpose of the present study was to determine the nature of the linkage of the Qa-2 antigen to the cell surface of preimplantation mouse embryos. It was found that all detectable Qa-2 antigen on the embryonic cell surface is sensitive to cleavage by PI-PLC and is therefore bound to the cell membrane by a GPI linkage. Furthermore, removal of Qa-2 antigen from the embryonic cell surface slows down the rate of development of preimplantation mouse embryos. These results suggest the likelihood that the Qa-2 antigen is the Ped gene product.