Expression of GlN1-Immunoreactive Glycoproteins during Development and Regeneration in Avian Peripheral Nerve

Abstract

Reactivity of the monoclonal antibody GlN1 was examined by immunocytochemistry and immunoblotting in quail and chick developing and regenerating wing nerves; its distribution is compared to that of HNK-1 which detects a carbohydrate epitope widely distributed in the nervous system. Reactivity was detected by immunofluorescence in cryostat sections, by a postembedding electron-microscopic immunogold technique and in immunoblots of nerve homogenates. From E11-E16, reactivity was detected in several large (120–260 kD) glycoprotein bands; and thereafter, principally, in 3 myelin-related glycoproteins (100, 26.5, and 21.5/19.5 kD). The HNK-1 carbohydrate epitope was detected in all these and in other bands permitting identification of the 100-kD moiety as the myelin-associated glycoprotein and the 26.5-kD protein as the P0 protein; the myelin-related 21.5/19.5-kD doublet appears as distinct, probable adhesion molecule(s). During development and regeneration, GlN1 reactivity detected by immunogold labeling of sections appeared first over the extracellular matrix, and later over thicker myelin sheaths. After transection, immunoreactivity in immunoblots was lost in distal stumps but reappeared with time; first in the larger (> 120 kD) moieties and then in the myelin-related bands, the same sequence observed in development. Electron-microscopic detection of both GlN1 and HNK-1 carbohydrate epitopes by immunogold labeling of resin-embedded sections is localized most consistently in the thicker (>0.3 µm) myelin sheaths of nerves from chicks at or after hatching. Immunoblots of mature fowl nerve tissues homogenized at various stages of preparation for electron microscopy (after fixation or after fixation and dehydration) show sustained immunoreactivity in the 21.5/19.5-kD bands, reduction or complete suppression in others, and evidence of immunoreactivity in high molecular weight, presumably cross-linked, constituents that remain in the stacking gel portion of the blots.

Immunoreactivity of PMP‐22, P0, and other 19 to 28 kDa glycoprotens in peripheral nerve myelin of mammals and fish with HNK1 and related antibodies

“…Furthermore, Smith and Perret (1986) reported that there was a glycoprotein of about this size (19 kDa) in bovine PNS myelin that did not react with their anti-PO antiserum. Also, Snyder et al (1990) demonstrated the presence of a 2 1 . 3 19.5 kDa glycoprotein in developing chicken and quail PNS myelin that reacted with HNKl and the related GIN1 monoclonal antibody, but not with an anti-PO antiserum, Similarly, our results in this paper show that there is a 21 kDa glycoprotein in cat myelin that reacts strongly with HNKl , but does not react with any of the monoclonal or polyclonal anti-PO antibodies tested.…”

“…The presence of peripheral nerve glycoproteins in the 19 to 28 kDa range in some species that react with HNK 1 and related anti-carbohydrate antibodies has now been reported by many investigators (Inuzuka et al, 1984;Nobile-Orazio et al, 1984;Noronha et al, 1986;Bollensen and Schachner, 1987;Bollensen et al, 1988;Burger et al, 1990;Snyder et al, 1990;van den Berg et al, 1990;Uyemura and Kitamura, 1991;Field et al, 1992). However, attempts to identify the reactive glycoproteins have led to variable interpretations.…”