Presence and release of SR-17 (chromogranin B(586-602)) in the porcine splenic nerve and its enzymatic degradation by CD26/dipeptidyl peptidase IV

Using the pig splenic nerve as a model, we investigated the proteolytic processing of porcine chromogranin B (CgB) during its axonal transport. An ELISA was developed for SR-17 (CgB(586-602)), a novel CgB-derived peptide, originally found in the adrenal medulla. The results demonstrate that CgB is processed in an early stage during its axonal transport. Immunohistochemical data, based on a rabbit anti-SR-17 antiserum, show that the spleen CgB/SR-17 is exclusively present in the nerve endings. No SR-17 immunoreactivity (IR) was found in splenocytes. We also provide evidence that SR-17 is co-released with noradrenaline (NA) upon electrical stimulation of the splenic nerve. Its release is frequency-dependent and strongly enhanced in the presence of the alpha-blocking agent phentolamine. In addition, we show that the new CgB-peptide can serve as a substrate for the lymphocyte surface glycoprotein CD26, also known as dipeptidyl peptidase IV (DPP IV), generating a new peptide ER-15 (CgB(588-602)).     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

Proteolytic processing of egg-laying hormone-related precursors in Aplysia. Identification of peptide regions critical for biological activity

The atrial gland of the marine mollusk Aplysia californica is an exocrine organ that expresses at least three genes belonging to the egg-laying hormone (ELH) family. In order to study the post-translational processing of the ELH-related gene products in the atrial gland and how it compares to the bag cells, peptides were isolated from the atrial gland and chemically characterized. The A- and B-related precursors were each cleaved in vivo to yield several major and minor peptides including peptides A and B and the ELH-related peptide complexes that caused egg laying. About 13% of the peptide complexes were further enzymically processed by the atrial gland to yield smaller fragments, which included A-AP.A-ELH-(15-36), A-AP.[Ala27]A-ELH-(15-36), and A-AP.[Gln23,Ala27]A-ELH-(16-36), where A-AP is an acidic peptide encoded by the A- and B-related genes and A-ELH is an ELH-related peptide encoded by the A gene. These processed peptide fragments were not active in an egg-laying bioassay, indicating that retention of the 14-residue NH2-terminal segment of the A-ELH-related sequence, or some portion thereof, was critical for the induction of egg laying. Other characterized peptides included two novel 13-residue NH2-terminal peptides, A-NTP and B-NTP, representing residues 22-34 of the A and B precursors, respectively. These two peptides occurred adjacent to the signal peptide region in each precursor, and their characterization established the site of signal peptide cleavage to be the Ser21-Gln22 peptide bond of each precursor. Intermediate peptide fragments (A-NTP-peptide A and B-NTP-peptide B) were also identified indicating that there was a specific ordering in the cleavage of peptide bonds during posttranslational processing. Finally, a new 55-residue atrial gland peptide was also isolated that was not a part of any ELH-related precursor characterized to date.     

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.     

Isolation and structure of the second of two major peptide products from the precursor to an anglerfish peptide homologous to neuropeptide Y

The peptide hormone recently isolated from anglerfish endocrine pancreas (aPY) (Andrews, P. C., Hawke, D., Shively, J.E., and Dixon, J.E. (1985) Endocrinology 116, 2677-2681), is a member of a family of peptide hormones which includes pancreatic polypeptide, neuropeptide Y, and the gut peptide YY. A 30-residue carboxyl-terminal fragment of the precursor to aPY has been purified from anglerfish endocrine pancreas in two steps using both classical chromatographic methods and reversed-phase high pressure liquid chromatography. It was identified by sequence homology with the analogous peptide from human preproneuropeptide Y. The sequence was found by Edman degradation and fast atom bombardment mass spectrometry to be SSPEEAVAWLLFKADPSQDIEPRLDDDNAW. The high yield of this fragment (6.5 nmol . g-1) is similar to that previously reported for aPY (7.9 nmol . g-1) and suggests that it is a major product of pro-aPY processing. The data indicate that pro-aPY is proteolytically processed into two major products: the 37-residue aPY and the 30-residue carboxyl-terminal fragment.     

Global profiling of proteolytically modified proteins in human metastatic hepatocellular carcinoma cell lines reveals CAPN2 centered network

Proteolysis affects every protein at some point in its life cycle. Many biomarkers of disease or cancer are stable proteolytic fragments in biological fluids. There is great interest and a challenge in proteolytically modified protein study to identify physiologic protease-substrate relationships and find potential biomarkers. In this study, two human hepatocellular carcinoma (HCC) cell lines with different metastasis potential, MHCC97L, and HCCLM6, were researched with a high-throughput and sensitive PROTOMAP platform. In total 391 proteins were found to be proteolytically processed and many of them were cleaved into persistent fragments instead of completely degraded. Fragments related to 161 proteins had different expressions in these two cell lines. Through analyzing these significantly changed fragments with bio-informatic tools, several bio-functions such as tumor cell migration and anti-apoptosis were enriched. A proteolysis network was also built up, of which the CAPN2 centered subnetwork, including SPTBN1, ATP5B, and VIM, was more active in highly metastatic HCC cell line. Interestingly, proteolytic modifications of CD44 and FN1 were found to affect their secretion. This work suggests that proteolysis plays an important role in human HCC metastasis.     

Facilitated glucose transporter of human erythrocyte: proteolytic mapping of the [3H]cytochalasin B photoaffinity-labeled transporter polypeptide

The human erythrocyte D-glucose transporter is an integral membrane glycoprotein with an heterogeneous molecular mass spanning a range 45-70 kDa. The protein structure of the transporter was investigated by photoaffinity labeling with [3H]cytochalasin B and fractionating the labeled transporter according to molecular mass by preparative SDS-polyacrylamide gel electrophoresis. Each fraction was digested with either papain or S. aureus V8 proteinase, and the labeled proteolytically derived peptide fragments were compared by SDS polyacrylamide gel electrophoresis. Papain digestion yielded two major peptide fragments, of approx. molecular mass 39 +/- 2 and 22 +/- 2 kDa; treatment with V8 proteinase resulted in two fragments, with mass of 24 +/- 2 and 15 +/- 2. Proteolysis of each transporter fraction produced the same pattern of labeled peptide fragments, irrespective of the molecular mass of the original fractions. The binding characteristics of [3H]cytochalasin-B-labeled transporter to Ricinis communis agglutinin lectin was examined for each transporter molecular mass fraction. It was found that higher-molecular-mass fractions of intact transporter had a 2-fold greater affinity for the lectin than lower-molecular-mass fractions (i.e., 67 kDa greater than 45 kDa fraction). However, proteolytically derived labeled peptide fragments from each fraction had minimal affinity for the lectin. These results suggest that the labeled peptide fragments have been separated from the glycosylated regions of the parent transporter protein. The present findings indicate that, although transporter proteins have an apparently heterogeneous molecular mass, some regions of the protein share a common peptide. Furthermore, the glycosylated regions appear to be located some distance from the [3H]cytochalasin-B-labeled site(s).     

Differential localization of processed fragments of Plasmodium falciparum serine repeat antigen and further processing of its N-terminal 47 kDa fragment

The serine repeat antigen (SERA) of Plasmodium falciparum is a blood stage malaria vaccine candidate. It has been shown that 120 kDa SERA was proteolytically processed into N-terminal 47 kDa fragment (P47), central 56 kDa fragment (P56) that was further converted to 50 kDa (P50), and C-terminal 18 kDa fragment (P18). Here, we have examined the processing of SERA and the localization of its processed fragments by using mouse antibodies directed against recombinant proteins corresponding to different domains of SERA. Western blot analysis showed that all the processing events occurred inside parasitized erythrocytes at the stage just prior to the schizont rupture, that P47 was further processed into two 25 kDa fragments and that the two fragments, which were linked to P18 through disulfide bonds, were associated with the merozoite. In contrast, P50 was completely shed into culture medium and absent from the merozoite. This observation was further supported by the results of indirect immunofluorescence assay. These results could account for the findings that antibodies against P47 were inhibitory to the parasite growth in vitro but those against P50 were not. Finally, we demonstrated that the further processing of P47 is allelic type-dependent. The results of the present study would help in vaccine designing based on SERA.     

Secretion of somatostatin by Saccharomyces cerevisiae. Correct proteolytic processing of pro-alpha-factor-somatostatin hybrids requires the products of the KEX2 and STE13 genes

Somatostatin is a 14-amino-acid peptide hormone that is proteolytically excised from its precursor, prosomatostatin, by the action of a paired-basic-specific protease. Yeast (Saccharomyces cerevisiae Mat alpha) synthesizes an analogous peptide hormone precursor, pro-alpha-factor, which is proteolytically processed by at least two separate proteases, the products of the KEX2 and STE13 genes, to generate the mature bioactive peptide. Expression in yeast of recombinant DNAs encoding hybrids between the proregion of alpha-factor and somatostatin results in proteolytic processing of the chimeric precursors and secretion of mature somatostatin. To determine if the chimeras were processed by the same enzymes that cleave endogenous pro-alpha-factor, the hybrid DNAs were introduced into kex2 and ste13 mutants, and the secreted proteins were analyzed. Expression of the pro-alpha-factor-somatostatin hybrids in kex2 mutant yeast resulted in secretion of a high molecular weight hyperglycosylated precursor. No mature somatostatin was secreted, and there was no proteolytic cleavage at the Lys-Arg processing site. Similarly, in ste13 yeast, only somatostatin molecules containing the (Glu-Ala)3 spacer peptide at the amino terminus were secreted. Our results demonstrate that in yeast processing mutants, the behavior of the chimeric precursors with respect to proteolytic processing was exactly as that of endogenous pro-alpha-factor. We conclude that the same enzymes that generate mature alpha-factor proteolytically process hybrid precursors. This suggests that structural domains of the proregion rather than the mature peptide are recognized by the processing proteases.     

Measurements of chromogranin B can serve as a complement to chromogranin A

OBJECTIVE: CgA has been shown to be an excellent marker for neuroendocrine tumours. However, there are two major drawbacks with CgA measurements; elevated levels are common in patients with decreased renal function and in patients on treatment with proton pump inhibitors. These problems are not seen with CgB measurements. We have recently presented the development of 13 region-specific radioimmunoassays for measurements of CgB. A region-specific assay was identified, which measured higher concentrations of CgB than the other assays and seemed to be very useful as a marker for neuroendocrine tumours. The aim of the present study was therefore to further explore the diagnostic potential of this assay in the clinical management of patients with neuroendocrine tumours. METHODS: Measurements of CgB with two methods were compared with CgA in plasma samples from patients investigated for neuroendocrine tumours (N=86), patients with decreased renal function (N=35) and patients on treatment with proton pump inhibitors (N=29). RESULTS: The diagnostic sensitivity for the new CgB assay was almost as good as that for CgA. Furthermore, with CgB measurements we could avoid the falsely elevated levels of CgA found in patients with decreased renal function and treatment with proton pump inhibitors. CONCLUSIONS: We conclude that the new CgB assay can serve as a complement to CgA measurements as an important tumour marker for neuroendocrine tumours.     

Human milk provides peptides highly stimulating the growth of bifidobacteria.

The large intestine of breast-fed infants is colonized predominantly by bifidobacteria, which have a protective effect against acute diarrhea. In this study we report for the first time the identification of human milk peptides that selectively stimulate the growth of bifidobacteria. Several bifidogenic peptides were purified chromatographically from pepsin-treated human milk and identified as proteolytically generated fragments from the secretory component of the soluble polyimmunoglobulin receptor and lactoferrin; both of these proteins exhibit antimicrobial effects. Hydrolysis of the identified peptides with the gastrointestinal proteases pepsin, trypsin and chymotrypsin did not lead to the loss of bifidogenic activity, indicating their potential function in vivo. Sequential comparison revealed a similar structural motif within the identified peptides. A correspondingly designed small peptide (prebiotic lactoferrin-derived peptide-I, PRELP-I) was found to stimulate the growth of bifidobacteria as effectively as the native peptides. The combination of antimicrobial and bifidobacterial growth stimulatory activity in human milk proteins leads to highly specific compounds capable of regulating the microbial composition of infants' large intestine.     

Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol

Pseudomonas exotoxin (PE) was incubated with cells and extracts analyzed for processed fragments. PE was proteolytically cleaved to produce a N-terminal 28-kDa and a C-terminal 37-kDa fragment, the latter being composed of a portion of domain II and all of domain III (the ADP-ribosylating domain). Cleavage was evident at 10 min after toxin addition and endosome preparations contained the processed fragments. Initially, the two fragments were linked by a disulfide bond. Subsequently, the 37-kDa fragment was reduced and translocated to the cytosol where it inactivated protein synthesis. Cytosol from toxin-treated cells was greatly enriched in the 37-kDa fragment. The 37-kDa fragment appears to be essential for toxicity since mutant PE molecules that do not produce this fragment, or cannot deliver it to the cytosol, fail to kill cells.     

A novel protease obtained from FBS-containing culture supernatant,that processes single chain form hepatocyte growth factor to two chain form in serum-free culture

The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.     

Immunohistochemical localization of chromogranin A and B in endocrine cells of the alimentary tract of the adult lizard Podarcis sicula

The distribution, argyrophilia, and the possible amine/peptide co-localizations in endocrine cells immunoreactive (IR) to antisera against chromogranin A (CgA) and chromogranin B (CgB) in the alimentary tract of the lizard Podarcis sicula have been investigated using novel monoclonal antibodies. Many CgA-IR and CgB-IR cells were found in the tract, except in the distal small intestine. Almost all chromogranin-IR cells (Cgs-IR) were also argyrophilic with parallel intensity. Some CgA-IR and CgB-IR cells did not display co-localized amines or peptides. CgA or CgB or both were found co-localized, with some local differences, in almost all serotonin-IR, histamine-IR, substance P-IR and gastric peptide tyrosine tyrosine (PYY)-IR cells. Moreover, both Cgs were co-localized only in some somatostatin-IR cells, whereas neurotensin-IR, gastrin/cholecystokinin-IR, pancreatic polypeptide-IR and intestinal PYY-IR cells did not show any co-localization with Cgs. The presence of Cgs in the endocrine cells was heterogeneous with regard to the complex interrelationship with their amine/peptide content. Consequently, Cgs cannot be considered as universal markers of all endocrine cell types.     

A 68 residue N-terminal fragment of pro-atrial natriuretic peptide is a monomeric intrinsically unstructured protein

The mature pro forms of the cardiac natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are proteolytically processed to their active hormone forms (28 and 32 residues, respectively) and N-terminal (NT)-pro fragments (68 and 76 residues, respectively). Far-ultraviolet circular dichroism (UV CD), 1D and 2D-homonuclear nuclear magnetic resonance (NMR), size exclusion-high performance liquid chromatography (SE-HPLC) and analytical ultracentrifuge sedimentation equilibrium (AUCSE) data are obtained for NT-proANP. CD data showed a large negative molar ellipticity for NT-proANP of -14,800° cm(2)/dmol at 199-200 nm. The intensity of the 1D-(1)H NMR spectra in the amide region for NT-proANP was very low and confined to ~8-8.6 ppm. Furthermore, cross-correlation resonance peaks were absent in the corresponding 2D-(1)H NOE spectra for NT-proANP in this region. The elution peak for this fragment from a G2000SW size-exclusion column was 20.4'; myoglobin (~17 K) was also eluted at 20.4'. No higher molecular weight oligomers were evident in the AUCSE experiments for NT-proANP. Collectively, the physical data demonstrate that NT-proANP, like NT-proBNP, is primarily a disordered, monomeric protein. Lastly, we compare the predictions from two in silico metaserver disorder algorithms, MeDor and MetaPrDOS, to the experimental data.     

Lassa virus glycoprotein signal peptide displays a novel topology with an extended endoplasmic reticulum luminal region

Lassa virus glycoprotein C (GP-C) is translated as a precursor (preGP-C) into the lumen of the endoplasmic reticulum (ER) and cotranslationally cleaved into the signal peptide and immature GP-C before GP-C is proteolytically processed into its subunits, GP-1 and GP-2, which form the mature virion spikes. The signal peptide of preGP-C comprises 58 amino acids and contains two distinct hydrophobic domains. Here, we show that each hydrophobic domain alone can insert preGP-C into the ER membrane. Furthermore, we demonstrate that the native signal peptide only uses the N-terminal hydrophobic domain for membrane insertion, exhibiting a novel type of a topology for signal peptides with an extended ER luminal part, which is essential for proteolytic processing of GP-C into GP-1 and GP-2.     

Role of Slit proteins in the vertebrate brain.

Diffusible chemorepellents play a major role in guiding developing axons towards their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptors and their secreted ligand Slits. Three distinct slit genes (slit1, slit2 and slit3) and three distinct robo genes (robo1, robo2 and rig-1) have been cloned in mammals. In collagen gel co-cultures, Slit1 and Slit2 can repel and collapse olfactory axons. However, there is also some positive effect associated with Slits, as Slit2 stimulates the formation of axon collateral branches by NGF-responsive neurons of the dorsal root ganglia (DRG). Slit2 is a large ECM glycoproteins of about 200 kD, which is proteolytically processed into 140 kD N-terminal and 55-60 kD C-terminal fragments. Slit2 cleavage fragments appear to have different cell association characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. This suggested that the different fragments might have different functional activities in vivo. We have begun to explore these questions by engineering mutant and truncated versions of hSlit2 representing the two cleavage fragments, N- and C-, and the uncleavable molecule and examining the activities of these mutants in binding and functional assays. We found that an axon's response to Slit2 is not absolute, but rather is reflective of the context in which the protein is encountered.     

Cathepsin L is involved in proteolytic processing of the Hendra virus fusion protein

Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F(0)) to the active F(1) + F(2) disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F(0) into F(1) and F(2) fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.     

Presence and release of the chromogranin B-derived secretolytin-like peptide KR-11 from the porcine spleen

Chromogranin B (CgB) is a major matrix protein in secretory granules/large dense-cored vesicles and a precursor for smaller peptides. In an earlier study, we have identified a secretolytin-like peptide (KR-11, pCgB(637-647)) from porcine chromaffin granules. Further evidence is presented here to show the processing of chromogranin B to this peptide during axonal transport in the splenic nerve and its release in the spleen upon various conditions of stimulation. Immunohistochemical staining showed that in the porcine spleen chromogranin B and NPY completely colocalize in nerve fibres and varicosities in blood vessel walls and trabeculae, and along the loose network of smooth muscle cells in the red pulp, as identified by their alpha-smooth muscle cell actin content. No antibacterial activity was found for the porcine secretolytin-like peptide, KR-11. The change of one amino acid residue (Thr-->Asn) in the porcine homologous fragment of secretolytin appears to be responsible for its loss of antibacterial activity.     

Cleavage of Ig-Hepta at a "SEA" module and at a conserved G protein-coupled receptor proteolytic site

Ig-Hepta is a member of a new subfamily of the heptahelical receptors and has an unusually long N terminus extending toward the extracellular side of the plasma membrane. Pulse-chase experiments in 293T cells using antisera specifically recognizing its N- and C-terminal regions demonstrated that Ig-Hepta is core-glycosylated cotranslationally and proteolytically processed into a two-chain form in the endoplasmic reticulum, followed by maturation of oligosaccharide chains and dimerization. The cleavage occurs at two highly conserved sites: one in a "SEA" module (a module first identified in sperm protein, enterokinase, and agrin) near the N terminus and the other in the stalk region preceding the first transmembrane span, generating approximately 20-, 130-, and 32-kDa fragments. The latter two remain tightly associated non-covalently even after cleavage as revealed by immunoprecipitation of native and myc-tagged Ig-Hepta constructs that were transiently expressed in 293T cells. The dimer consisting of four chains, (130 kDa + 32 kDa)(2), is linked by disulfide bonds. A fusion protein of the extracellular domain of Ig-Hepta and the Fc domain of immunoglobulin was found to be a good substrate of the processing enzymes and used for determining the exact cleavage sites in the SEA module and juxtamembrane stalk region.