(Received for publication, July 31, 1995; and in revised form, November 28, 1995)
From the
The complete deduced primary structure of mouse brain testican has been established from cDNA cloning. The cDNA encodes a polypeptide of 442 amino acids belonging to the proteoglycan family. The mouse brain testican core protein is 95% identical to its human testicular counterpart. In situ hybridization investigations revealed that mouse testican mRNA is mainly present in a subpopulation of pyramidal neurons localized in the CA3 area of the hippocampus. An immunocytochemical approach, with antibodies directed against an overexpressed chimeric antigen, produced in bacterial systems, showed that testican is associated with the postsynaptic region of these pyramidal neurons. Testican includes several putative functional domains related to extracellular or pericellular proteins associated with binding and/or regulatory functions. On the basis of its structural organization and its occurrence in postsynaptic areas, this proteoglycan might contribute to various neuronal mechanisms in the central nervous system.
Proteoglycans (PGs) ()have been demonstrated to be
involved in mechanisms such as cell adhesion, cell migration, and cell
proliferation (Ruoslahti, 1989; Ruoslahti and Yamaguchi, 1991; Wight et al., 1992).
Proteoglycans of the nervous system have been reviewed recently (Margolis and Margolis, 1993; Lander, 1993; Oohira et al., 1994). Chondroitin sulfate and keratan sulfate proteoglycans are mainly found in the extracellular space, whereas heparan sulfate proteoglycans are predominantly located on the cell surface (Margolis and Margolis, 1989). A developmentally regulated expression of PGs in various areas of the central nervous system has been described (Krueger et al., 1992; Rauch et al., 1992; Maeda et al., 1992; Carey et al., 1992; Stipp et al., 1994). PGs play a pivotal role in the growth and morphology of neurons (Cole and McCabe, 1991; Oohira et al., 1991; Faissner et al., 1994). GAGs have been shown to modulate axonal outgrowth and dendrite elongation (Damon et al., 1988; Lafont et al., 1994). The protein cores of PGs have also been proposed to participate in these events (Perris and Johansson, 1990; Iijima et al., 1991).
We have recently characterized a human testicular PG, named ``testican'' on the basis of its first identification in testes (Alliel et al., 1993). The analysis of a mouse multiple tissue Northern blot with human testican cDNA probes revealed that hybridizing transcripts are restricted to mouse brain. In order to define a murine model suitable for functional characterization of testican in brain, the complete primary structure of mouse brain testican was deduced from cDNA cloning experiments. The human and mouse testican protein cores were compared, and their multidomain structure is discussed and connected with potential biological functions. In situ hybridization showed that testican transcripts are mainly detected in pyramidal cells of the hippocampus. Immunohistochemical localization studies further indicated that testican is localized in the postsynaptic area of these neurons. A contribution of testican to a postsynaptic complex of these cells is suggested.
Figure 2: Cloning, nucleotide, and deduced amino acid sequences of mouse testican. a, the establishment of a 4343-bp composite cDNA sequence encoding mouse testican was established by the analysis of nine cDNA overlapping clones. A and B denote the human testican cDNA probes used in this study; M and T in the MB6 clone denote the probes used for the in situ hybridization experiments. The shaded box corresponds to the coding region. E corresponds to restriction enzyme sites for EcoRI. Scale bar, 0.5 kb. b, the deduced amino acid sequence is shown below the nucleotidic sequence. Single-letter code is used, and numbers indicate the positions of nucleotide or amino acid residues starting at the initiation codon. The first 21 amino acids fit with the von Heijne (1986) consensus for a leader peptide. The putative cleavage site is shown by an arrow. Cytidine 10, in boldface type, was identified as a guanosine in CSA6. A putative polyadenylation signal (nucleotides 1953-1958), also described in human testican, is in italic type. These sequence data are available from EMBL under accession number X92864.
Nucleic acid sequence comparison between mouse and human testicans was performed following the DIAGON program (Staden, 1982), using the CITI-2 (Paris, France) sequence analysis software.
Figure 1:
Northern
blot analysis of testican mRNA in mouse tissues. Poly(A) mRNA (2 µg/lane) from heart (lane 1), brain (lane 2), spleen (lane 3), lung (lane 4),
liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), and testis (lane 8) were hybridized with
human testican cDNA probe B (see Fig. 2). Arrows show
the position of the size markers: 1.35, 2.4, 4.4, 7.5, and 9.5
kb.
Three positive clones, MB4, MB6, and MB8, were selected during a
first screening of the gt11 mouse brain cDNA library, using human
testican probe B. The two largest EcoRI restriction fragments
from these mouse cDNA clones, which hybridized either with human probe
A or B, were used as selective probes during a second screening of the
gt11 mouse brain library. Four independent clones (CSA4, CSA5,
CSA6, and CSA10) were selected. The larger 3` EcoRI
restriction fragment in the CSA5 clone did not hybridize with any human
probe used in this study; it allowed us to select two additional clones
(TCS5-1 and TCS5-2) during a last screening (Fig. 2a).
The initiation codon is followed by a 1326-bp open reading frame, which codes for a polypeptide of 442 amino acids. The mouse nucleotide coding sequence and its deduced protein sequence are 90 and 95% identical to those of human testican, and 60% of the amino acid substitutions are conservative (Fig. 3a). Serine residues 386 and 391, whose counterparts are the GAG attachment sites in human testican, are conserved. The main difference between both testicans is a Glu-Val-Glu tripeptide insertion at positions 63-65 in mouse brain testican.
Figure 3: Comparison between mouse and human testicans. a, comparison between mouse and human testican deduced amino acid sequences. Lines between residues refer to identity, and dots refer to homologous amino acids (A/G/S/T, D/E/N/Q, I/L/V, Y/F/W, and R/K/H). A probable processing site, based upon statistical data, is indicated by an arrow. Putative glycosaminoglycan attachment sites are indicated by arrowheads. b, dot matrix plot analysis of mouse and human testicans nucleic acid sequences. The analysis was plotted by the DIAGON program (Staden, 1982), with a score of 8 and a window size of 9. Numbering for mouse and human cDNA testicans begins at nucleotide 1 and 295, respectively. The comparison extends over 3200 nucleotides. Solid lines represent the coding regions of human (horizontal) and mouse (vertical) testican.
A dot matrix plot comparing human and mouse testican cDNAs indicated that transcript homologies extend beyond the coding sequence both in the 5` and 3` regions (Fig. 3b). A putative polyadenylation site (ATTAAA), conserved in both mouse and human transcripts, was identified at position 1953. The use of this polyadenylation site may give rise to minor mRNA, with a shortened 3` region.
Figure 4:
Distribution of testican transcripts in
brain, analyzed by in situ hybridization. MB6-M (nucleotides
627-1098) and MB6-T (nucleotides 1099-2030) cDNA probes are EcoRI restriction fragments from MB-6 clone (see Fig. 2a). a-d, macroscopic images of
testican mRNA distribution on serial parasagittal to sagittal brain
sections of a 15-day-old mouse after hybridization with the S-labeled mouse MB6-M cDNA probe. Lane 1, cortex; lane 2, corpus callosum; lane 3, striatum; lane
4, thalamus; lane 5, hypothalamus; lane 6,
hippocampus. e-j, in situ localization of
testican mRNA on brain sections of a 15-day-old mouse brain at the
cellular level using either the
S-labeled MB6-M (e-h) or MB6-T (i-j) cDNA probes. e and f, the images correspond to the insert shown in b. Numerous silver grains are present on the neurons of the
hippocampus CA3 area; in contrast the granule cells of the dentate
gyrus (DG) are clearly not labeled. g and h,
the images correspond to the insert shown in d. Intense
labeling in the CA3 is observed; the proximal cortex (Co) is
less labeled, and the corpus callosum (CC) shows a background
distribution of silver grains. i and j, view of
pyramidal cells in the CA3 hippocampal at higher magnification.
Autoradiograms were photographed under epipolarization light (e, g, i) or bright field illumination (f, h, j) after thionine blue staining with
a LEICA DMRB microscope. Scale bars in e and f, 25 µm; scale bars in h and j, 50 µm.
Figure 5: Immunolocalization of testican in mouse brain. Transverse vibratome sections of 21-day-old mouse brain immunostained with PTC9 antiserum (1:500). Diffuse and punctate immunolabeling is observed in the area of the pyramidal neurons of the hippocampus. In the CA3 area the punctate labeling can be correlated to the spines of the dendrites (arrows) originating from pyramidal neurons (a, b). In other brain regions, i.e cerebellum (c) and cortex (d), only a faint punctate labeling (arrows) is noticed. In cerebellum (c), the staining is clearly limited to the molecular layer (ML). P, Purkinje cells; GL, granular layer. Scale bars, 50 µm.
Figure 6: Immunoelectron microscopic localization of testican. Testican localization in the postsynaptic portion of synapses present in the stratum lucidum of the hippocampus (a, b), cortex (c, d), and molecular layer of the cerebellum (e) of a 21-day-old mouse brain after incubation with PTC9 antiserum (1:500). The labeling (open arrows) is strong on the postsynaptic (Po) density and faint in the immediate cytoplasmic fluid. No labeling is present in the synaptic cleft. The corresponding presynaptic areas (Pr), clearly identified by the presence of synaptic vesicles, are also negative; not all the postsynaptic densities are immunostained (black arrow). Scale bars, 0.5 µm.
The composite sequence, established from nine mouse brain
cDNA clones, encodes a 442-amino acid polypeptide highly homologous to
the human testican protein core. Structural conservation around the so
far identified GAG attachment sites suggests that mouse brain testican
may be substituted by chondroitin sulfate and heparan sulfate, as shown
for testican from the human male reproductive organs (Bonnet et
al., 1992). Neither any other serine-glycine pairs, matching the
consensus sequence corresponding to GAG attachment sites (Bourdon,
1990), nor NXS or NXT N-linked glycosylation
consensus sequences are present within mouse or human testican deduced
protein sequences. The Northern blot analysis was indicative of a
brain-restricted distribution of testican transcripts in mice; however,
reverse transcriptase-polymerase chain reaction also allowed their
identification in testes. ()Human transcripts appeared more
broadly distributed, but, among 16 tested tissues, brain is one of the
three tissues where testican transcripts were mostly detected. (
)
The high conservation of mouse and human polypeptidic
structures led us to consider that functional elements reside within
the testican's protein core. Such conserved structures are
encountered in proteins involved in basic cell biological processes.
The distribution of the proline residues, which account for 5.4% of the
total amino acids, is not random. The PCPCLPEPEPLKP
peptide contains 25% of the total proline residues clustered in a
region corresponding to less than 3% of the protein. The human
counterpart of this peptide, despite a variation in the proline residue
distribution, shows the same features. The tetrapeptide GKSL is present
at positions 172-175 and in the palindromic peptide LSKGKSL
(amino acids 326-332). In mouse testican, the supernumerary
tripeptide
EVE is inserted in a pseudopalindromic
structure,
WNRFRDEVEDDYFRNW. The corresponding peptide in
human testican allows us to determine a
WNRFXD(1-4)DXFRNW palindromic sequence (X is a nondefined amino acid, and 1-4 denotes the number of
spacing amino acids) whose biological significance, if any, is not
known yet. Testican contains a cluster of glutamic acid and aspartic
acid residues, which account for 40% of the 63 C-terminal residues.
This acidic region includes a stretch of 9 consecutive acidic amino
acids. A similar stretch is also encountered in other PGs: in the
-amyloid precursor protein (Kang et al., 1987), in
claustrin (Burg and Cole, 1994), in versican (Zimmermann and Ruoslahti,
1989), and in brevican (Yamada et al., 1994). Such a stretch
may be involved in the binding of cationic substances. The QKLSK
pentapeptide, located in the upstream vicinity of the CWCV domain, is
highly reminiscent of the NKISK sequence, which has been shown to be of
importance for the binding of decorin to fibronectin (Schmidt et
al., 1991).
The mouse brain testican, as its human testicular counterpart (Alliel et al., 1993), is a multidomain protein that shares substantial similarities with proteins involved in adhesion, migration, and cell proliferation. Some of these domains have been identified in proteins involved in neural development, synaptogenesis, and synaptic transmission. The counteradhesive neural molecules QR1 and SC1, either associated with neurite outgrowth and synapse formation (Guermah et al., 1991) or expressed during neural postnatal development (Johnston et al., 1990), contain testican's osteonectin-like domains. A 45-amino acid sequence related to Kazal-type protease inhibitor domains (Apostol et al., 1993) exists as a single copy in QR-1 and SC-1, and is tandemly repeated in follistatin (Hemmati-Brivanlou et al., 1994). Besides its implication for the gonadal function, follistatin-activin interaction has been suggested to contribute to the maintenance of nerve survival (Schubert et al., 1990), to be involved in the control of neurosecretory neuron activity (Sawchenko et al., 1988), and to participate in neural differentiation (Hashimoto et al., 1992). A wide protease inhibitory effect has been demonstrated for a recombinant rat agrin (Biroc et al., 1993), a protein that contains nine Kazal-type domains (Ferns and Hall, 1992) and plays a role in acetylcholine receptors clustering around the neuromuscular junctions. Agrin, which can be substituted by GAG chains (Tsen et al., 1995), has been proposed to be an interactive partner of neural cell adhesion molecules that may regulate a variety of cell adhesion processes, including synaptogenesis. A 46-amino acid motif, the CWCV domain, has been identified in various proteins associated to the basement membrane and epithelial cell surface (Alliel et al., 1993). It is of interest to notice that this interspecies-conserved domain in the IGF-BP family (Shimasaki et al., 1991) is assumed to contribute to growth factor binding (Huhtala et al., 1986). Heparin-like GAGs have been proposed to act as local regulators of IGF/IGF-BP complexes (Arai et al., 1994). IGFs are known to influence neurotransmission in the hippocampus and regulation of synaptic transmission occurs through modulation of transmitter receptors in the postsynaptic membrane (Araujo et al., 1989). Thus testican is a molecule whose structure contains peptidic motifs and can be substituted by polysaccharides encountered in contributors to extracellular matrix organization and in regulators of neuralization and neurotransmission.
The spatial immunolabeling distribution of testican in mouse brain correlates well with the in situ hybridization results. Glial cells appear to be devoid of testican. Among the neuron-enriched brain regions, testican is mainly distributed on the postsynaptic densities of neurons occurring mostly in the CA3 area. The staining is concentrated at the postsynaptic side of the plasma membrane of the dendrite. This observation and structural features of testican suggest that this PG is likely to interact with membrane-bound components at the postsynaptic nerve terminals. Testican contains neither an hydrophobic domain, except for the N-terminal putative signal peptide, nor a C-terminal sequence that could be glypiated (Gerber et al., 1992). The ability of the core protein of some brain PGs to interact with neurons via neural cell adhesion molecules has been recently proposed as a regulatory mechanism for cell adhesion and cell migration in the central nervous system (Grumet et al., 1993; Friedlander et al., 1994; Maurel et al., 1994; Grumet et al., 1994). The testican transcripts and protein core are mainly distributed within the pyramidal cells in the hippocampus CA3 region where glypican is also present (Karthikeyan et al., 1994). On the basis of its structure, mimicking a set of proteins sharing diverse binding abilities, it is tempting to hypothesize that testican modulates the binding of ligands to target cells. It may play a crucial role in specialized neurons, in a brain area where receptors for growth factors have been identified (Fayen et al., 1992; Marks et al., 1991; Breese et al., 1991).
Among the proteoglycan protein cores so far depicted, none can be structurally related to testican. This molecule can thus be considered as a new member of the brain proteoglycan family. The spatial distribution of testican in a restricted area of the brain, together with its appearance in the postsynaptic region, is an argument for its potential contribution to receptor activity, neuromodulation, synaptic plasticity, or even neurotransmission.