Cell biology of polysialic acid

The unusual carbohydrate polysialic acid (PSA), attached uniquely to neural cell adhesion molecule (NCAM) through a developmentally regulated process, modulates neural cell interactions. Major advances in the past two years have increased our understanding of PSA biosynthesis and regulation. Of particular interest is the cloning of the genes encoding polysialyltransferases (PSTs) and the finding that a single enzyme is able to confer polysialylation to NCAM. The electrical activity of neurons and transmembrane signalling are probably major players in controlling both PSA biosynthesis and its expression at the cell surface. A direct causal relationship between PSA expression and activity-induced synaptic plasticity has been reported.     

Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol-anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.     

Population subdivision among desert bighorn sheep (Ovis canadensis) ewes revealed by mitochondrial DNA analysis

Modulation of neural cell adhesion molecule polysialylation (NCAM PSA) state has been proposed to underlie morphofunctional change associated with consolidation of memory in the rodent, and its age-dependent decline to be related to impaired cognitive function. To establish whether this may be a human correlate of cognitive decline, we determined the age-dependent expression of PSA in the human hippocampal dentate gyrus using postmortem tissue derived from individuals who exhibited no obvious neuropathology. As in the rodent, PSA immunoreactivity in the 5-month human infant was associated mainly with a population of granule-like cells and their mossy fibre axons. Cell numbers were maximal during the first 3 years of life but declined by an order of magnitude between the second and third decades and remained relatively constant thereafter and was restricted to the granule cell layer/hilar border. In contrast to the rodent, diffuse immunostaining was observed in the inner molecular layer; however, as development advanced, this became relocated to the outer molecular layer from 2 years of age onwards. In addition, numerous polysialylated hilar neurons became evident at 2-3 years of age and remained constant until the eighth decade of life. These findings suggest NCAM polysialylation to play a crucial developmental role within a period concluding with adolescence, and that an attenuated NCAM PSA-mediated neuroplasticity continues throughout the human lifespan. The importance of the developmental phase of NCAM PSA expression in the emergence of schizophrenia is discussed.     

Age-related changes in neural cell adhesion molecule (NCAM) isoforms in the mouse telencephalon

The concentrations of different polypeptide isoforms of the neural cell adhesion molecule NCAM were examined in telencephalic and brainstem-cerebellar tissue from groups of young (3 months) and old (25 months) mice. Antibodies against chick brain NCAM were used in immunoblot analyses to quantify 180 (NCAM180) and 140 (NCAM140) kDa NCAM forms in mouse brain samples containing equal amounts of protein. Telencephalic homogenates from the older group exhibited 37% and 31% less NCAM180 and NCAM140 immunoreactivity, respectively, when compared with homogenates from the younger animals. Brainstem-cerebellar homogenates, however, did not express such age-related changes in the two NCAM isoforms. Age-related changes in isoforms labeled by the anti-NCAM antibodies were not evident in synaptic plasma membranes. NCAM180:NCAM140 ratios were 2- to 3-fold greater in the synaptic membranes vs. homogenates for both age groups. These data suggest that expression levels of NCAM180 and NCAM140 are selectively impaired with aging in the telencephalon, whereas the synaptic contents of these molecules appear to be stably regulated.     

Kv1.1 channel antisense attenuates learning and modulation of dentate polysialylated NCAM

The distribution and modulation of neural cell adhesion molecule polysialylation state (NCAM PSA) and the consequence of antisense inactivation of the Kv1.1 potassium channel was investigated following avoidance learning in mice. PSA immunoreactivity was most notable on cells at the inner denate border and in cortical layer II. Task acquisition resulted in a significant 30% transient increase in the frequency of dentate polysialylated neurons at the 12 h post-training time. In contrast, animals pretreated with the Kv1.1 antisense oligonucleotide exhibited both attenuated recall avoidance latencies and polysialylated cell frequency. As Kv1.1 is enriched on the dendrites of these granule-like cells, the attenuated polysialylation response is considered secondary to NCAM-mediated events during their transient synapse production in the 6-8 h post-training period.     

Protein kinase C phosphorylates the "a" forms of plasma membrane Ca2+ pump isoforms 2 and 3 and prevents binding of calmodulin

Phosphorylation by protein kinase C of the "a" and "b" variants of plasma membrane Ca2+ pump isoforms 2 and 3 was studied. Full-length versions of these isoforms were assembled and expressed in COS cells. Whereas the "a" forms were phosphorylated easily with PKC, isoform 2b was phosphorylated only a little, and isoform 3b was not phosphorylated at all. Phosphorylation of isoforms 2a and 3a did not affect their basal activity, but prevented the stimulation of their activity by calmodulin and their binding to calmodulin-Sepharose. This indicated that phosphorylation prevented activation of these isoforms by preventing calmodulin binding. Based on these results, phosphorylation of the pump with PKC would be expected to increase free intracellular Ca2+ levels in those cells where isoforms 2a and 3a are expressed.     

A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.     

Expression of regulated cardiac troponin I in Escherichia coli

The study of the functional effects of troponin isoform changes would be greatly aided by the development of a strategy permitting protein engineering and mutational analysis. To assess the role of troponin isoforms in regulating myofibrillar ATPase activity, we have expressed rat cardiac troponin I (cTnI) in E. coli and purified the protein to near homogeneity. We utilized the inducible expression vector pGEX-KG to create a glutathione-S-transferase fusion protein which can be cleaved with thrombin. Approximately 6 mg of cTnI can be purified from 1 l of culture. Ca2+Mg2+ ATPase activity was measured using the bacterially synthesized cTnI and the remaining components of the regulated actomyosin complex (troponin T, troponin C, tropomyosin, actin, and myosin) purified to homogeneity from mammalian hearts. In the presence of free Ca2+ ranging from 10(-2) to 10(-8) M, bacterially synthesized cTnI exhibits specific activity similar to that observed for control cTnI isolated from rat hearts. The bacterially synthesized protein is capable of stoichiometric phosphorylation and demonstrates appropriately regulated specific activity. These results establish the feasibility of using bacterial expression to study functional consequences of changes in expression of troponin isoforms.     

Polarity of epithelial cells of the liver. Cellular and molecular mechanisms, and pathologic changes

Smooth muscle contraction is primarily regulated not only by changes in cytosolic Ca2+ concentrations ([Ca2+]i) but also by changes in the force/[Ca2+]i ratio. The use of membrane-permeabilization technique facilitated demonstration of an increase in the level of force at constant [Ca2+]i (Ca2+ sensitization). It was clarified that Rho-associated kinase (Rho-kinase) is a novel mediator of Ca2+ sensitization of the smooth muscle contraction, by introducing the recombinant catalytic domain of Rho-kinase into the cytosol of vascular smooth muscle permeabilized with beta-escin. This review article focuses on novel mechanisms, by which activation of receptor-coupled G-protein(s) increases Ca2+ sensitivity of the contractile apparatus in smooth muscle: Rho-kinase and protein kinase C.     

Neural cell adhesion molecules, learning, and memory in the domestic chick

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick forebrain is a site of recognition memory for the learning process of imprinting. The results reported here demonstrate that neural cell adhesion molecules (NCAMs) play a time-dependent role in this recognition memory. Dark-reared chicks were trained, tested, and assigned a preference score as a measure of learning. Chicks with high preference scores were designated good learners and those with lower preference scores, poor learners. Controls were untrained. Tissue was removed, 9.5 hr or 24 hr after training, from the left and right IMHV, hyperstriatum accessorium, and posterior neostriatum. Three major NCAM isoforms (180, 140, and 120 kDa) were assayed. At 24 hr only, there was in left IMHV significantly more NCAM (for each isoform) in good learners than in the other 2 groups, and also a significant correlation between the amounts of NCAM and preference scores for all isoforms; the amount predicted by each regression line at preference score 50 (no learning) did not differ significantly from the mean value for untrained controls. There were no learning-related effects in either the hyperstriatum accessorium or the posterior neostriatum.     

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension-Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.     

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.     

The expression of plasma membrane Ca2+ pump isoforms in cerebellar granule neurons is modulated by Ca2+

Plasma membrane Ca2+ ATPase (PMCA) pump isoforms 2, 3, and 1CII are expressed in large amounts in the cerebellum of adult rats but only minimally in neonatal cerebellum. These isoforms were almost undetectable in rat neonatal cerebellar granule cells 1-3 days after plating, but they became highly expressed after 7-9 days of culturing under membrane depolarizing conditions (25 mM KCl). The behavior of isoform 4 was different: it was clearly detectable in adult cerebellum but was down-regulated by the depolarizing conditions in cultured cells. 25 mM KCl-activated L-type Ca2+ channels, significantly increasing cytosolic Ca2+. Changes in the concentration of Ca2+ in the culturing medium affected the expression of the pumps. L-type Ca2+ channel blockers abolished both the up-regulation of the PMCA1CII, 2, and 3 isoforms and the down-regulation of PMCA4 isoform. When granule cells were cultured in high concentrations of N-methyl-D-aspartic acid, a condition that increased cytosolic Ca2+ through the activation of glutamate-operated Ca2+ channels, up-regulation of PMCA1CII, 2, and 3 and down-regulation of PMCA4 was also observed. The activity of the isoforms was estimated by measuring the phosphoenzyme intermediate of their reaction cycle: the up-regulated isoforms, the activity of which was barely detectable at plating time, accounted for a large portion of the total PMCA activity of the cells. No up-regulation of the sarcoplasmic/endoplasmic reticulum calcium pump was induced by the depolarizing conditions.     

NCAM140 interacts with the focal adhesion kinase p125(fak) and the SRC-related tyrosine kinase p59(fyn)

Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59(fyn) in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59(fyn) from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59(fyn) did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. pp60(c-)src, a tyrosine kinase that promotes neurite growth on the neuronal cell adhesion molecule L1, did not interact with any NCAM isoform. Whereas p59(fyn) was constitutively associated with NCAM140, the focal adhesion kinase p125(fak), a nonreceptor tyrosine kinase known to mediate integrin-dependent signaling, became recruited to the NCAM140-p59(fyn) complex when cells were reacted with antibodies against the extracellular region of NCAM. Treatment of cells with a soluble NCAM fusion protein or with NCAM antibodies caused a rapid and transient increase in tyrosine phosphorylation of p125(fak) and p59(fyn). These results suggest that NCAM140 binding interactions at the cell surface induce the assembly of a molecular complex of NCAM140, p125(fak), and p59(fyn) and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.     

Intracellular location, temporal expression, and polysialylation of neural cell adhesion molecule in astrocytes in primary culture

Neural cell adhesion molecules (NCAMs) constitute a group of cell surface glycoproteins that control cell-cell interactions and play important morphoregulatory roles in the developing and regenerating nervous system. NCAMs exist in a variety of isoforms differing in the cytoplasmic domain and/or their content in sialic acid. The highly sialylated form (PSA-NCAM) is expressed by neurons, whereas it is believed that the less sialylated NCAM forms are synthesised by astrocytes. Moreover, little is known about the molecular sequence of the events that contribute to its expression at the cell surface. Here we report that during the proliferation of cortical astrocytes, at 4 days in primary culture, these cells expressed PSA-NCAM as well as NCAM 180. Then, during cell differentiation these isoforms progressively disappeared and the NCAM 140 became predominant. By immunofluorescence and immunocytochemistry studies we also show that PSA-NCAM and NCAM are first observed in small cytoplasmic spots or vesicles, located in or near the Golgi apparatus, as demonstrated by their co-localization with labelled wheat germ agglutinin (WGA) in this cell organelle. Thereafter, immunostained cytoplasmic NCAM gradually disappeared and became detectable at the cell surface of differentiating astrocytes. We also describe for the first time sialyltransferase activity in these cells and report that the levels of this activity correlated with the decrease in PSA-NCAM expression during the differentiation of astrocytes. These results will contribute to our understanding of the PSA and NCAM intracellular transport pathways and their expression at the cell surface. Moreover, the presence of PSA-NCAM in astrocytes suggests their possible role in nerve branching, fasciculation, and synaptic plasticity.     

Molecular characterization of eukaryotic polysialyltransferase-1

Polysialic acid (PSA) is a dynamically regulated product of post-translational modification of the neural cell adhesion molecule, NCAM. Presence of the large anionic carbohydrate modulates NCAM binding properties and, by increasing the intercellular space, influences interactions between other cell surface molecules. PSA expression underlies cell type- and developmental-specific alterations and correlates with stages of cellular motility. In the adult, PSA becomes restricted to regions of permanent neural plasticity and regenerating neural and muscle tissues. Recent data implicate its important function in spatial learning and memory, and in tumour biology. Here we describe the molecular characterization of polysialyltransferase-1, the key enzyme of eukaryotic PSA synthesis. In reconstitution experiments, the newly cloned enzyme induces PSA synthesis in all NCAM-expressing cell lines. Our data therefore represent convincing evidence that the polycondensation of alpha-2,8-linked sialic acids in mammals is the result of a single enzymatic activity and provide a new basis for studying the functional role of PSA in neuro- and tumour biology.     

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures

We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.     

A role for polysialic acid in neural cell adhesion molecule heterophilic binding to proteoglycans

The neural cell adhesion molecule (NCAM) is known to participate in both homophilic and heterophilic binding, the latter including mechanisms that involve interaction with proteoglycans. The polysialic acid (PSA) moiety of NCAM can serve as a negative regulator of homophilic binding, but indirect evidence has suggested that PSA can also be involved in heterophilic binding. We have examined this potential positive role for PSA in terms of the adhesion of PSA-expressing mouse F11 cells and chick embryonic brain cells to substrates composed of the purified heparan sulfate proteoglycans agrin and 6C4. This adhesion was specifically inhibited by polyclonal anti-NCAM Fab antibodies, monoclonal anti-PSA antibodies, PSA itself, and enzymatic removal of either PSA or heparan sulfate side chains. By contrast, the adhesion was not affected by chondroitinase, and cell binding to laminin was not inhibited by any of these treatments. A specific NCAM-heparan sulfate interaction in this adhesion was further indicated by its inhibition with monoclonal anti-NCAM Fab antibodies that recognize the known heparin-binding domain of NCAM and with the HBD-2 peptide derived from this region, but not with antibodies directed against other regions of the protein including the homophilic binding region. Together, the results suggest that PSA can act in vitro either as a receptor in NCAM heterophilic adhesion or as a promoter of binding between heparan sulfate proteoglycans and the NCAM heparin-binding domain.