Henk Zwiers

B-50 (GAP-43): biochemistry and functional neurochemistry of a neuron-specific phosphoprotein.

Abstract: The biochemistry and functional neurochemistry of the synaptosomal plasma membrane phosphoprotein B‐50 (GAP‐43) are reviewed. The protein is putatively involved in seemingly diverse functions within the nervous system, including neuronal development and regeneration, synaptic plasticity, and formation of memory and other higher cognitive behaviors. There is a considerable amount of information concerning the spatial and temporal localization of B‐50 (GAP‐43) in adult, fetal, and regenerating nervous tissue but far less is known about the physical chemistry and biochemistry of the protein. Still less information is available about posttranslational modifications of B‐50 (GAP‐43) that may be the basis of neurochemical mechanisms that could subsequently permit a variety of physiological functions. Hence, consideration is given to several plausible roles for B‐50 (GAP‐43) in vivo, which are discussed in the context of the cellular localization of the protein, significant posttranslational enzymes, and regulatory proteins, including protein kinases, phosphoinositides, calmodulin, and proteases.

Evidence for a Single Protein Kinase C‐Mediated Phosphorylation Site in Rat Brain Protein B‐50

Abstract: The neuronal protein B‐50 may be involved in diverse functions including neural development, axonal regeneration, neural plasticity, and synaptic transmission. The rat B‐50 sequence contains 226 amino acids which include 14 Ser and 14 Thr residues, all putative sites for phosphorylation by calcium/phospholipid‐dependent protein kinase C (PKC). Phosphorylation of the protein appears to be a major factor in its biochemical and possibly its physiological activity. Therefore, we investigated rat B‐50 phosphorylation and identified a single phosphorylated site at Ser41. Phosphoamino acid analysis eliminated the 14 Thr residues because only [32P]Ser was detected in an acid hydrolysate of [32P]B‐50. Staphylococcus aureus protease peptide mapping produced a variety of radiolabeled [32P]B‐50 products, none of which had the same molecular weights or HPLC retention times as several previously characterized fragments. Indirect confirmation of the results was provided by differential phosphorylation of major and minor forms of B‐60 that have their N‐termini at, or C‐terminal to, the Ser41 residue and are the major products of specific B‐50 proteolysis. Only those forms of B‐60 that contained the Ser41 residue incorporated phosphate label. The results are discussed with reference to the substrate requirements for B‐50 phosphorylation by PKC and the proposed structure of the B‐50 calmodulin binding domain.

Distribution of growth-associated protein, B-50 (GAP-43) in the mammalian enteric nervous system

The presence of the growth-associated protein, B-50 (also known as GAP-43) was investigated in the adult mammalian enteric nervous system. The small intestine of rat, ferret and human was examined by immunohistochemistry. Dense B-50-like immunoreactivity was localized in nerves throughout the wall of the rat, ferret and human small intestine, notably in the myenteric and submucous plexuses, where in the ferret ileum it co-localized with vasoactive intestinal polypeptide-immunoreactive fibre groups. Material with the biochemical and immunological characteristics of rat B-50 was extracted from the rat ileum. In-situ hybridization demonstrated that enteric neurons express B-50. These findings are consistent with a role for B-50 in the documented plasticity of the adult enteric nervous system.

TELEOST ISOTOCIN RECEPTOR : STRUCTURE, FUNCTIONAL EXPRESSION, MRNA DISTRIBUTION AND PHYLOGENY

A cDNA encoding a receptor for the oxytocin‐related peptide isotocin has been identified by screening a lambda gt11 library constructed from poly(A)+ RNA of the hypothalamic region of the teleost Catostomus commersoni. The probe used was obtained by PCR amplification of white sucker genomic DNA using degenerate primers based on conserved sequences in the mammalian receptor counterparts. The full‐length cDNA specifies a polypeptide of 390 amino acid residues that displays the typical hydrophobicity profile of a seven transmembrane domain receptor and which exhibits greatest similarity to mammalian oxytocin receptors. Oocytes that express the cloned receptor respond to the application of isotocin by an induction of membrane chloride currents indicating that it is coupled to the inositol phosphate/calcium pathway. The isotocin receptor (ITR) can also be activated by vasotocin, mesotocin, oxytocin and Arg‐vasopressin, although these have lower potencies than isotocin. ITR‐encoding mRNA has been detected in brain, intestine, bladder, skeletal muscle, lateral line, gills and kidney indicating that this receptor may mediate a variety of physiological functions.

The dental amalgam mercury controversy — inorganic mercury and the CNS; genetic linkage of mercury and antibiotic resistances in intestinal bacteria

Mercury (Hg) vapor exposure from dental amalgam has been demonstrated to exceed the sum of all other exposure sources. Therefore the effects of inorganic Hg exposure upon cell function in the brain and in the intestinal bacteria have recently been examined. In rats we demonstrate that ADP-ribosylation of tubulin and actin brain proteins is markedly inhibited, and that ionic Hg can thus alter a neurochemical reaction involved with maintaining neuron membrane structure. In monkeys we show that Hg, specifically from amalgam, will enrich the intestinal flora with Hg-resistant bacterial species which in turn also become resistant to antibiotics.

ADP-Ribosylation of the Neuronal Phosphoprotein B-50/GAP-43

Abstract: The neuronal phosphoprotein B‐50/GAP‐43 is associated with growth and regeneration within the nervous system and its posttranslational status can be correlated with its cellular localization during growth and regeneration. Recently, B‐50 has been shown to interact with certain G protein subunits. Regulation of G protein‐mediated signal transduction may involve ADP‐ribosylation in vivo. In the present study we have demonstrated that B‐50 is a substrate for endogenous ADP‐ribosyltransferases. The results are discussed with respect to the possible interaction of B‐50 with G proteins, but also with regard to the posttranslational modification of B‐50 by all major regulatory mechanisms that act at, or through, the neuronal membrane.

The growth-associated neuronal phosphoprotein B-50: improved purification, partial primary structure, and characterization and localization of proteolysis products.

A reversed phase HPLC procedure is reported that has allowed the separation of the growth-associated, kinase C substrate protein B-50 [previously purified by isoelectric focussing (IEF)] into three components (1-, m- and rB-50). The minor form 1B-50 (probably a proteolysis product) gave a 24-residue N-terminal amino acid sequence, but the major and possibly native form (mB-50) (and also rB-50 which is probably formed during IEF) appeared to be N-terminally blocked. HPLC also separated B-60, the major proteolysis product of B-50, into three components, and the N-terminal sequence of the major B-60 was determined. HPLC peptide mapping of SAP digests of the various B-50 and B-60 protein confirmed their close relationship, and four SAP generated fragments also afforded sequence data. The amino acid sequences obtained (1B-50, B-60 and fragments) are all found in the recently predicted (based on nucleotide sequencing) B-50/GAP43 sequence (226 amino acids), further confirming the identity of B-50 and GAP43, and helping clarify the relationship of B-60 (starting at position 41 of the predicted sequence) to B-50. Correlation of amino acid analyses, SAP fragment data, and the predicted sequence provided additional information on the length of the translated products, including evidence that the N-terminus of the major (blocked) form of B-50 starts at position 1 (Met) of the predicted sequence.

ADP-Ribosylation of Brain Neuronal Proteins Is Altered by In Vitro and In Vivo Exposure to Inorganic Mercury

Abstract: ADP‐ribosylation is an essential process in the metabolism of brain neuronal proteins, including the regulation of assembly and disassembly of biological polymers. Here, we examine the effect of HgCl2 exposure on the ADP‐ribosylation of tubulin and actin, both cytoskeletal proteins also found in neurons, and B‐50/43‐kDa growth‐associated protein (B‐50/GAP‐43), a neuronal tissue‐specific phosphoprotein. In rats we demonstrate, with both in vitro and in vivo experiments, that HgCl2 markedly inhibits the ADP‐ribosylation of tubulin and actin. This is direct quantitative evidence that HgCl2, a toxic xenobiotic, alters specific neurochemical reactions involved in maintaining brain neuron structure.

Peptides in the gastrointestinal tract in human immunodeficiency virus infection

The presence of immunoreactivity to the neuronal phosphoprotein B-50 and the peptides bombesin, calcitonin gene-related peptide, galanin, neurotensin, neuropeptide Y, somatostatin, substance P, and vasoactive intestinal polypeptide was examined in biopsy specimens from the duodenum and rectum of human immunodeficiency virus (HIV)-seropositive and HIV-seronegative male homosexual patients. The distribution of B-50 and the peptides was correlated with HIV serology, number of CD4+ lymphocytes, and the presence of HIV in biopsy culture. There was a very low incidence of enteric pathogens in both groups of patients. It was found that HIV-seropositive patients had a greater incidence of abnormal patterns of immunoreactivity (reduced intensity and/or density of innervation) in enteric nerves and enteroendocrine cells than HIV-seronegative patients. A reduction of substance P immunoreactivity was significantly correlated with reduced CD4+ lymphocyte count and HIV status; a similar trend was also seen for somatostatin and vasoactive intestinal polypeptide. Using B-50 as a marker, it was found that both groups of patients had altered patterns of immunoreactivity in rectal nerves. The findings of this study suggest that some of the clinical symptoms associated with HIV infection may be caused by a specific HIV enteropathy that influences enteric nerve and/or enteroendocrine cell function by altering the density of peptide immunoreactivity.

Monoclonal Antibody NM2 Recognizes the Protein Kinase C Phosphorylation Site in B-50 (GAP-43) and in Neurogranin (BICKS)

Abstract: Mouse monoclonal B‐50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B‐50 (GAP‐43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted with peptide fragments of rat B‐50 containing the unique protein kinase C (PKC) phosphorylation site at serine‐41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B‐50 (34–52). To narrow down the epitope domain, synthetic B‐50 peptides were tested in ELISAs. In contrast to NM6, NM2 immunoreacted with B‐50 (39–51) peptide, but not with B‐50 (43–51) peptide or a C‐terminal B‐50 peptide. Preabsorption by B‐50 (39–51) peptide of NM2 inhibited the binding of NM2 to rat B‐50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B‐50 during endogenous phosphorylation of synaptosomal plasma membrane proteins. Preabsorption of NM2 by B‐50 (39–51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B‐50 and neurogranin. Therefore, NM2 may be a useful tool in physiological studies of the role of PKC‐mediated phosphorylation and calmodulin binding of B‐50 and neurogranin.