Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes.

Posttranslational modifications and intracellular transport of the D2-cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A) and 131,000-158,000 (B) were synthesized using radiolabeled precursors in cultured neurons. A and B were found to contain only N-linked complex oligosaccharides, and both polypeptides appeared to be polysialated as determined by [14C]mannosamine incorporation and precipitation with anti-polysialic acid antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination by photoactivation employing a hydrophobic labeling reagent. In rat forebrain explant cultures, D2-CAM was synthesized as four polypeptides: A (195,000 Mr), B (137,000 Mr), C (115,000 Mr), and a group of polypeptides in the high molecular weight region (HMr) between 250,000 and 350,000. Peptide maps of the four polypeptides yielded similar patterns. Biosynthesis of C and HMr increased with age, relative to A and B. A and B were sulfated in embryonic brain, however, sulfation was not noticeable at postnatal ages. Phosphorylation, on the other hand, of A and B was observed at all ages examined. We suggest that D2-CAM function may be modified during development by changes in the relative synthesis of the different polypeptides, as well as by changes in their glycosylation and sulfation.

Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells.

D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing chase times the Mr of both molecules increased to 187,000-201,000 (A) and 137,000-158,000 (B). These were similar to the sizes of D2-CAM labeled with [14C]glucosamine, [3H]fucose and [14C]mannosamine, indicating that the higher Mr species are glycoproteins. In the presence of tunicamycin, which specifically blocks the synthesis of high mannose cores, Mr were reduced to 175,000 (A) and 124,000 (B). Newly synthesized A and B are susceptible to degradation by endo-beta-N-acetyl-glucosaminidase H, which specifically degrades high mannose cores, but they are resistant to such degradation after 150 min of posttranslational processing. Hence, we deduce that A and B are initially synthesized with four to five high mannose cores which are later converted into N-linked complex oligosaccharides attached to asparagine residues. However, no shift of [35S]methionine radioactivity between A and B was detected with different pulse or chase times, showing that these molecules are not interconverted. Thus, our data indicate that the neuronal D2-CAM glycoproteins are derived from two mRNAs.

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C.

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.

Characterization of soluble forms of NCAM.

Neural cell adhesion molecule (NCAM) has been described as a family of membrane glycoproteins. However, soluble NCAM immunoreactivity has long been recognized. We here show that soluble NCAM is composed of two quantitatively major polypeptides of Mr 180,000 and 115,000 and two minor components of Mr 160,000 and 145,000. Soluble NCAM was immunochemically identical to membrane NCAM, was polysialylated and carried the HNK-1 epitope. It only constituted 0.8% of total NCAM in newborn rat brain. Soluble NCAM appeared in neuronal cell culture medium 15-30 min after the start of synthesis preceding accumulation of membrane-associated NCAM on the cell surface. This indicates that soluble NCAM contains a secreted component.

Matrigel enhances myotube development in a serum-free defined medium.

Previously reported serum-free defined media for muscle cell culture require supplementation with hormones, purified growth factors or attachment factors. This report describes a culture system that enhances embryonic chick, skeletal muscle cell growth and differentiation in a serum-free defined medium, without added specialized trophic factors. Myoblasts adhered more to and proliferated more rapidly on a reconstituted basement membrane substrate, Matrigel, than on rat-tail collagen. Matrigel contains several basement membrane attachment molecules which apparently obviate the need for added purified attachment factors. Matrigel also appeared to play a trophic role in subsequent development by enabling the serum-free growth of myotubes which suggests that Matrigel mediates the cellular interaction of growth or attachment factors. Collagen, on the other hand, did not support serum-free myotube growth. Supplementation of defined medium with increasing levels of horse serum enhanced total protein in myotubes grown on both substrates; protein was higher in Matrigel cultures for each medium tested. The serum-free defined medium supported complete morphological differentiation of myotubes grown on Matrigel and maintained myotube cultures up to 22 days. Fibroblast proliferation was higher in cultures on collagen in defined medium with high serum levels, but was virtually eliminated in cultures on Matrigel in serum-free defined medium. The culture system described supports the differentiation of embryonic muscle cells in a simple, serum-free defined medium, thus providing an in vitro model of developing myotubes which should be particularly useful for studies of regulation mediated by extracellular factors.

Molecular forms of the cholinesterases inside and outside muscle endplates.

Individual endplates were micro-dissected from chicken fast-twitch muscle, and the molecular forms of acetylcholinesterase and of pseudocholinesterase therein, identified by their sedimentation coefficients, were analysed directly. The forms actually present at the endplate, and those that are non-synaptic, were established. This analysis was also extended to muscle of the chicken with inherited muscular dystrophy, showing altered distributions of these forms.

Changes in glucose 6-phosphate dehydrogenase activity in developing embryonic chick skeletal muscle and spinal cord.

Glucose 6-phosphate dehydrogenase (G6PDH) activity was examined in the developing embryonic chick in brachial and lumbar spinal cord and pectoral and leg muscle. Enzyme activity was generally highest at the earliest stage examined, embryonic day 5. The developmental profiles for G6PDH activity in the two muscles were similar: a sharp initial decrease occurred between days 5 and 9, with relatively low levels present by day 18; peaks of G6PDH activity at days 12 and 16 were more prominent in leg muscle. Similar levels of G6PDH were also detected in spinal cord with the developmental profile in the brachial spinal cord resembling that seen in muscle. In lumbar spinal cord, initial G6PDH activity was lower than in brachial spinal cord; the developmental profile, however, resembled that seen in the brachial spinal cord, with an initial drop in enzyme activity seen between days 5 and 7. Neural regulation of G6PDH activity in mature muscle is believed to repress enzyme synthesis. The drop in G6PDH activity observed in embryonic spinal cord and muscle between days 5 and 9 coincides with the initiation of functional neuromuscular contacts. Hence, the normal regulation of G6PDH during embryonic development may involve the repression of G6PDH in spinal cord neurons and muscle, possibly effected by their interaction.

Fibre types in chicken skeletal muscles and their changes in muscular dystrophy.

1. Five major fibre types in chicken skeletal muscles are recognized, based upon their histochemical and morphological characteristics. A classification of these which is readily related to a commonly used classification of mammalian muscle fibre types is given.2. Seven muscles of the chicken were analysed in recognizing this range of fibre types. The proportions of the different types in each of these were determined. In some cases a gradient of fibre type composition exists across a single muscle.3. Measurements of muscle contraction were used in defining tonic muscles, which contain two fibre types. It was shown that in addition to the anterior latissimus dorsi (a.l.d.), previously well known to be a tonic muscle, two other muscles, the plantaris and the adductor profundus, are of the same class, but differ subtly from the a.l.d. in certain features. Gross red colouration is not a useful diagnostic feature of slow muscles, since the tonic adductor profundus, for example, is white.4. Fibres similar histochemically to mammalian type I (slow-twitch) occur in some of the avian twitch muscles investigated. These are oxidative in character, and despite the fact that they are multiply innervated we suggest that these are avian slow-twitch fibres.5. The patterns of cholinesterases found in a skeletal muscle correspond to its fibre type composition, with regard to both the concentrations and the proportions of the multiple forms of enzyme present. The distinctive patterns of those forms of acetylcholinesterase in the different fibre types are described.6. The fibre type composition is changed by inherited muscular dystrophy in a characteristic manner. This change has so far been found (at the earlier stages of the disease) only in the muscles with a predominance of type II B fibres in the normal chicken. Pathological changes within the fibres occur selectively in the type II B fibres, but there are exceptions to this and the effect can be greatly modified by the type of neighbouring fibres.

A developmental study of the biosynthesis of the neural cell adhesion molecule.

The neural cell adhesion molecule (N-CAM) is a glycoprotein found in neurons, glial cells and muscle cells. In this report we describe developmental changes in biosynthesis of N-CAM polypeptides in rat forebrain explant cultures. N-CAM was synthesized as the following polypeptides: HMr (Mr between 250,000 and 350,000), A (200,000 Mr), B (135,000 Mr) and C (115,000 Mr). The biosynthetic pattern of N-CAM polypeptides changed during development: the biosynthesis of HMr and C increased relative to A and B. N-CAM biosynthesis decreased 100-fold from embryonic day 17 to postnatal day 25; N-CAM turnover decreased 350-fold during the same period. N-CAM polysialylation and sulfatation decreased markedly with age, whereas phosphorylation seemed to be constant during development. Only polypeptides A and B were phosphorylated, whereas A, B and C were sulfated. A was more sulfated and phosphorylated than B. It is concluded that the above described modulations of N-CAM may be of importance in the developmental regulation of cell-cell adhesion.

Molecular forms of acetylcholinesterase and pseudocholinesterase in chicken skeletal muscles: their distribution and change with muscular dystrophy.

Chicken muscles offer several significant advantages for the use of cholinesterase as a marker of nerve-muscle interactions. A series of molecular forms of chicken muscle acetylcholinesterase (AChE), and likewise of pseudocholinesterase (psi ChE), has been defined. The form of AChE inside the endplates of fast-twitch muscle is H2c (20 S), with a collagenous tail. The same is true for psi ChE. The changes in these forms in the muscle with embryonic development, with muscle fibre-type composition and under the influence of inherited muscular dystrophy, are described quantitatively.

Comparison of the molecular forms of the cholinesterases in tissues of normal and dystrophic chickens.

The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (psiChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (Hc) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and psiChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or psiChE in a slow-tonic muscle. In the pectoral of PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the pesChE pattern remains abnormal. Muscle psiChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of psiChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and Hc. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or psiChE were found in cardiac or neural tissues from dystrophic chickens.

Regulation of NCAM by growth factors in serum-free myotube cultures.

Regulation of the neural cell adhesion molecule (NCAM) was examined in primary cultures of chick skeletal muscle grown in serum-free defined medium. Relative levels of NCAM (per microgram protein) increased 20-30% in myotubes grown on Matrigel, a reconstituted basement membrane preparation, compared to those grown on collagen; total NCAM levels on Matrigel were increased 40-55% due to the additional increase in total protein. A dose dependent increase in relative NCAM levels in myotubes grown on Matrigel in defined medium was observed with the addition of adsorbed horse serum, while relative NCAM levels in myotubes grown on collagen were unaffected by altering the serum concentration. Thus, extracellular matrix molecules and soluble factors exert trophic effects on myotube NCAM expression. Similar developmental changes in the expression of the different molecular size forms of NCAM occurred in myotubes grown on collagen and Matrigel: levels of 150K and 135K Mr forms decreased during development, while 125K remained prominent in older myotubes. Relative NCAM levels were specifically enhanced 11-26% by several factors: nerve growth factor, thyroxine, insulin-like growth factor II, dibutyryl cyclic AMP, veratridine (a sodium ion channel agonist), and nisoldipine (a calcium ion channel agonist). Total protein and overall myotube development in serum-free cultures were enhanced by fetuin, insulin-like growth factor II, acidic fibroblast growth factor, calcitonin gene-related peptide, dibutyryl cyclic AMP, and veratridine. Thus, changes in extracellular matrix, intracellular calcium, and sodium ions, as well as extracellular trophic factors, such as nerve growth factor, thyroxine, and insulin-like growth factor II, may regulate muscle NCAM expression during embryonic development.