Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibres in the penile cavernous tissue of green monkeys (Cercopithecus aethiops)

Summary The cavernous body of green monkeys contains many unmyelinated and few myelinated axons. The unmyelinated axons form terminals in the adventitia of the arteries, between trabecular muscle cells, in the interstitium, and close to endothelium cells of the sinuses. All terminals displayed predominantly small clear vesicles and very few large granular vesicles; small granular vesicles were not seen. However, in rabbit penises, terminals with many large granular vesicles are prominent. Immunohistochemistry (PAP technique) showed a dense network of VIP- and NPY-reactive fibres around the arteries and around trabecular muscles. The density of nerve fibres was particularly high around the subendothelial cushions of the helicine arteries. Double staining for NPY and VIP revealed that both peptides were colocalized. Immunocytochemistry (preembedding PAP technique) showed VIP- and NPY-reactivity in terminals with small clear vesicles; the reaction product was bound to the cytoplasmic face of different membrane types. Although the intracellular localization of the reaction product is probably due to artefactual displacement during preparation, the uniformity of the terminals questions the view that large and small granular vesicles in all species characterize peptidergic and noradrenergic terminals, respectively. The essential findings can be summarized as (1) a high degree of uniformity of nerve terminals, (2) colocalization of VIP and NPY, (3) heavy innervation of the subendothelial cushions of the helicine arteries, and (4) possible innervation of endothelial cells.     

The occurrence and distribution of certain polypeptides within the human carotid body

Summary Both carotid bodies from 26 patients coming to necropsy were fixed in 10% neutral buffered formalin and sections 4 m thick were stained for various peptides by use of the immunogold technique. The results show that the human carotid body contains met- and leu-enkephalin, substance P, vasoactive intestinal peptide (VIP), neurotensin and bombesin. The distribution of these six peptides within the carotid body differs. Thus met- and leu-enkephalin are both present predominantly within glomic chief cells but with a marked tendency to favour the dark variant of these cells. Substance P and VIP both show a weak immunoreactivity in comparison to the enkephalins and are present in all three variants of chief cell. Neurotensin shows the weakest immunoreactivity of all and is restricted to a few glomic chief cells in a minority of cases. Bombesin also shows a weak immunoreactivity in glomic chief cells but a strong reaction in glomic arteries and arterioles. In these vessels bombesin appears to be confined to smooth muscle cells in the media but we cannot say whether it is secreted by them or merely bound to receptor sites on their membranes. These findings are related to quantitative data on the concentration of peptides in the human carotid body from a previous paper with which we were associated.     

Neuropeptide Y: presence in sympathetic and parasympathetic innervation of the nasal mucosa

Summary The occurrence of neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in the sympathetic and parasympathetic innervation of the nasal mucosa was studied in various species including man. A dense network of NPY-immunoreactive (IR) fibres was present around arteries and arterioles in the nasal mucosa of all species studied. NPY was also located in nerves around seromucous glands in pig and guinea-pig, but not in rat, cat and man. The NPY-IR glandular innervation corresponded to about 20% of the NPY content of the nasal mucosa as revealed by remaining NPY content determined by radioimmunoassay after sympathectomy. These periglandular NPY-positive fibres had a distribution similar to the VIP-IR and PHI-IR nerves but not to the noradrenergic markers tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH). The NPY nerves around glands and some perivascular fibres were not influenced by sympathectomy and probably originated in the sphenopalatine ganglion where NPY-IR and VIP-IR ganglion cells were present. The venous sinusoids were innervated by NPY-positive fibres in all species except the cat. Dense NPY and DBH-positive innervation was seen around thick-walled vessels in the pig nasal mucosa; the latter may represent arterio-venous shunts. Double-labelling experiments using TH and DBH, and surgical sympathectomy revealed that the majority of NPY-IR fibres around blood vessels were probably noradrenergic. The NPY-positive perivascular nerves that remained after sympathectomy in the pig nasal mucosa also contained VIP/PHI-IR. The major nasal blood vessels, i.e. sphenopalatine artery and vein, were also densely innervated by NPY-IR fibres of sympathetic origin. Perivascular VIP-IR fibres were present around small arteries, arterioles, venous sinusoids and arterio-venous shunt vessels of the nasal mucosa whereas major nasal vessels received only single VIP-positive nerves. The trigeminal ganglion of the species studied contained only single TH-IR or VIP-IR but no NPY-positive ganglion cells. It is concluded that NPY in the nasal mucosa is mainly present in perivascular nerves of sympathetic origin. In some species, such as pig, glandular and perivascular parasympathetic nerves, probably of VIP/PHI nature, also contain NPY.     

Functional vasoactive intestinal polypeptide (VIP)-system in salt glands of the Pekin duck

Summary In saltwater-acclimated ducks with fully specialized supraorbital salt glands, intracarotid application of acetylcholine (5 nmoles/min/kg b.w.) or porcine vasoactive intestinal polypeptide (pVIP) (240 pmoles/min/kg b.w.) induced secretion from the salt glands at threshold conditions of secretory activity. pVIP-like immunoreactivity could be localized in fibers of the postganglionic secretory nerve ramifying throughout the glandular parenchyma. Both middle-sized arterioles and secretory tubules were innervated, and pVIP-immunoreactive varicose fibers formed peritubular baskets around the basal region of secretory tubules indicating direct innervation of the secretory tissue. pVIP-specific staining could be abolished by preabsorption of the antiserum with peptide extracts of salt-gland tissue. Synthetic pVIP and endogenous VIP from salt glands of the duck co-eluted on the HPLC system, suggesting structural similarity of the peptides. Membrane-binding studies with radioiodinated pVIP revealed the presence of high-affinity binding sites in salt-gland tissue. Affinities of unlabeled pVIP analogues to compete for these binding sites were as follows: pVIP > PHI > pVIP antagonist > secretin > pVIP (10–28) > chicken VIP (16–28). Peptide extracts of salt glands had affinities similar to pVIP. Binding sites could be localized mainly at the apical end of the radially arranged secretory tubules, as demonstrated by receptor autoradiography.It is concluded that, in addition to the classical parasympathetic transmitter acetycholine, VIP serves as neuromodulator/transmitter in cranial parasympathetic control of avian salt-gland secretion by acting on both the arteriolar network and the secretory tubules of the gland.     

Ontogeny of substance P-, CGRP-, and VIP-containing nerve fibers in the amphibian carotid labyrinth of the bullfrog,Rana catesbeiana

Summary The ontogeny of substance P, CGRP (calcitonin gene-related peptide), and VIP (vasoactive intestinal polypeptide) containing nerve fibers in the carotid labyrinth of the bullfrog, Rana catesbeiana, was examined by the peroxidase-antiperoxidase method. The time of appearance of these three peptides was different for each. First, CGRP fibers appeared in the wall of the carotid arch and external carotid arteries, and in a thin septum between these two arteries at an early stage of larval development (stage III). At stage V, substance P immunoreactive fibers appeared, and VIP fibers were detected at the early metamorphic stage (stage XXII). Up to the completion of metamorphosis, the number of these fibers remained low. From 1 to 5 weeks after metamorphosis, substance P, CGRP, and VIP fibers increased in number to varying degrees. By 8 weeks after metamorphosis, the distribution and abundance of these fibers closely resembled those of the adults. Some CGRP and VIP immunoreactive glomus cells were found at the stages immediately before and after the completion of metamorphosis. These findings suggest that substance P, CGRP, and VIP fibers during larval development and metamorphosis may be nonfunctional, and start to participate in vascular regulation only after metamorphosis. The transient CGRP and VIP in some glomus cells may be important for the development of the labyrinth, or may take part in vascular regulation through the close apposition of the glomus and smooth muscle cells (g-s connection).     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

Hypermotility induced by vasoactive intestinal peptide in the rat: its reciprocal action to cholecystokinin octapeptide

Intracerebroventricular administration of vasoactive intestinal peptide (VIP) shortened the duration of pentobarbital-induced sleep and produced significant hypermotility in the rat. Although hypermotility induced by methamphetamine was not potentiated by central administration of VIP, L-DOPA-induced hypermotility in pargyline-pretreated rats was markedly enhanced by VIP and this hypermotility was suppressed by simultaneous administration of cholecystokinin octapeptide (CCK-8) in a dose-related manner. Apomorphine-induced hypermotility was also potentiated by VIP. These results suggest that VIP may stimulate postsynaptic dopaminergic receptor, causing an increase in motility, and that a possible reciprocal interaction exists between VIP and CCK-8.     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.