A monoclonal antibody that blocks the activity of a neurite regeneration-promoting factor: studies on the binding site and its localization in vivo.

Abstract

Abstract. Work from several laboratories has identified a proteoglyean complex secreted by a variety of non-neuronal cells that can promote neurite regeneration when applied to the surface of culture dishes. Using a novel immunization protocol, a monoclonal antibody (INO) was produced that blocks the activity of this outgrowth-promoting factor (Matthew, W. D., and P. H. Patterson, 1983, Cold Spring Harbor Symp. Quant. Biol. 48:625-631). We have used the antibody to analyze the components of the active site and to localize the complex in vivo. INO binding is lost when the complex is dissociated; if its components are selectively reassociated, INO binds only to a complex containing two different molecular weight species. These are likely to be laminin and heparan sulfate proteoglycan, respectively. On frozen sections of adult rat tissues, INO binding is present on the surfaces of glial cells of the peripheral, but not the central, nervous system. INO also binds to the basement membrane surrounding cardiac and skeletal muscle cells, and binding to the latter greatly increases after denervation. In the adrenal gland and kidney, INO selectively reacts with areas rich in basement membranes, staining a subset of structures that are immunoreactive for both laminin and heparan sulfate proteoglycan. In general, the outgrowth-blocking antibody binds to areas known to promote axonal regeneration and is absent from areas known to lack this ability. This suggests that this complex, which is active in culture, may be the physiological substrate supporting nerve regeneration in vivo.oNs damaged in the peripheral nervous system (PNS) 1 of adult mammals have the capacity to regenerate over long distances. The prerequisites for successful neurite outgrowth include the presence of trophic factors, such as nerve growth factor (3), and/or an appropriate surface on which to grow (30). The molecular nature of the surfaces that allow or stimulate regeneration in vivo is not known. Experiments in culture, however, have delineated a number of candidate molecules that can promote neurite outgrowth under specific conditions. For instance, surfaces coated with laminin or fibronectin stimulate a much more rapid and extensive regeneration of neuronal processes than can serum-or collagen-coated plastic (2,4,9,24,29,31,39,44). The finding that laminin, in particular, provides an especially good substrate for outgrowth is of interest because a variety of non-neuronal cells secrete neurite regeneration-promoting factors (1,12,14,26), some of which are extracellular matrix complexes containing primarily laminin and heparan sulfate proteoglycan (HeSPG) (8,16,28).Since laminin is a component of a naturally occurring,1. Abbreviations used in this paper: CM, conditioned medium; CNS, central nervous system; DRG, dorsal root ganglion; HeSPG, heparan sulfate proteoglycan; PNS, peripheral nervous system. neurite-promoting complex, and purified laminin can stimulate outgrowth in culture, the question arises as to whether there is a role for the HeSPG compo...

Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies.

“…One corollary of these observations is that the neurite-promoting site of native laminin molecules does not seem to be significantly antigenic, antibody inhibition relying either on epitopes provided by the 440-kD A chain close to the site (as discussed for EHS laminin), or by more distant sites when the range of the blockade is increased by the use of secondary antibodies (17). Alternatively, it has been shown that a blocking epitope can be created by the association of laminin with other molecules (7).…”

Extracellular Matrix Molecules And Their Receptors: Functions In Neural Development

“…In almost all cases, LN expression appears to be transient, occurring at high levels during periods of axonal growth and, at least, in the central nervous system, diminishing later in development. Two notable exceptions are the continued expression of LN isoforms in the basal lamina of peripheral nerve (Chiu et al 1986) and the optic nerves of goldfish (Hopkins et al 1985, Liesi 1985, two favorable sites in the adult for either continued axonal growth or nerve regeneration. In contrast to studies on cell migration, surprisingly few attempts have been made to use antibodies or other reagents to interfere with axonal growth.…”

“…The INO antibody was isolated as an inhibitor of the activity of LN-containing neurite outgrowth factors (Matthew & Patterson 1983). It clearly binds an epitope associated with LN-heparan sulfate complexes (Chiu et al 1986). It also inhibits axonal extension on sections of the peripheral nerve where merosin, B1, and B2 are the major visible LN subunits (Sandrock & Matthew 1987b, Sanes et al 1990.…”

Relationship between sprouting axons, proteoglycans and glial cells following unilateral nigrostriatal axotomy in the adult rat

“…For example, addition of soluble HS disturbs growth of cockroach pioneer axons (Wang and Denburg, 1992) while scission with HS lyases a¡ects retinotectal path¢nding in Xenopus embryos (Walz et al, 1997). HSPG^laminin complexes also modulate neurite outgrowth in vitro including growth promotion and growth inhibition (Lander et al, 1985a,b;Chiu et al, 1986;Hantaz-Ambroise et al, 1987;Mathiessen et al, 1989). For example, whereas monoclonal antibodies to HSPG reduce neurite outgrowth on sciatic nerve in vitro (Sandrock and Matthew, 1985) a Schwannoma-derived HSPG also blocks the neurite-promoting activity of laminin (Muir et al, 1989).…”