Glycosylation of Cblns attenuates their receptor binding.

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo.

Comparison of the enzymatic and functional properties of three cytosolic carboxypeptidase family members.

Nna1 (CCP1) defines a subfamily of M14 metallocarboxypeptidases (CCP1-6) and is mutated in pcd (Purkinje cell degeneration) mice. Nna1, CCP4, and CCP6 are involved in the post-translational process of polyglutamylation, where they catalyze the removal of polyglutamate side chains. However, it is unknown whether these three cytosolic carboxypeptidases share identical enzymatic properties and redundant biological functions. We show that like Nna1, purified recombinant CCP4 and CCP6 deglutamylate tubulin, but unlike Nna1, neither rescues Purkinje cell degeneration in pcd mice, indicating that they do not have identical functions. Using biotin-based synthetic substrates, we established that the three enzymes are distinguishable based upon individual preferences for glutamate chain length, the amino acid immediately adjacent to the glutamate chain, and whether their activity is enhanced by nearby acidic amino acids. Nna1 and CCP4 remove the C-terminal glutamate from substrates with two or more glutamates, whereas CCP6 requires four or more glutamates. CCP4 behaves as a promiscuous glutamase, with little preference for chain length or neighboring amino acid composition. Besides glutamate chain length dependence, Nna1 and CCP6 exhibit higher k(cat)/K(m) when substrates contain nearby acidic amino acids. All cytosolic carboxypeptidases exhibit a monoglutamase activity when aspartic acid precedes a single glutamate, which, together with their other individual preferences for flanking amino acids, greatly increases the potential substrates for these enzymes and the biological processes in which they act. Additionally, Nna1 metabolized substrates mimicking the C terminus of tubulin in a way suggesting that the tyrosinated form of tubulin will accumulate in pcd mice.

A structural and functional analysis of Nna1 in Purkinje cell degeneration (pcd) mice.

The axotomy-inducible enzyme Nna1 defines a subfamily of M14 metallocarboxypeptidases, and its mutation underlies the Purkinje cell degeneration (pcd) mouse. However, the relationship among its catalytic activity, substrate specificities, and the critical processes of neurodegeneration/axon regeneration is incompletely understood. Here we used a transgenic rescue strategy targeting expression of modified forms of Nna1 to Purkinje cells in pcd mice to determine structure-activity relationships for neuronal survival and in parallel characterized the enzymatic properties of purified recombinant Nna1. The Nna1 subfamily uniquely shares conserved substrate-determining residues with aspartoacylase that, when mutated, cause Canavan disease. Homologous mutations (D1007E and R1078E) inactivate Nna1 in vivo, as does mutation of its catalytic glutamate (E1094A), which implies that metabolism of acidic substrates is essential for neuronal survival. Consistent with reports that Nna1 is a tubulin glutamylase, recombinant Nna1-but not the catalytic mutants-removes glutamate from tubulin. Recombinant Nna1 metabolizes synthetic substrates with 2 or more C-terminal glutamate (but not aspartate) residues (V(max) for 3 glutamates is ∼7-fold higher than 2 glutamates although K(M) is similar). Catalysis is not ATP/GTP dependent, and mutating the ATP/GTP binding site of Nna1 has no effect in vivo. Nna1 is a monomeric enzyme essential for neuronal survival through hydrolysis of polyglutamate-containing substrates.

Comparison of Cbln1 and Cbln2 functions using transgenic and knockout mice.

Cerebellin precursor protein 1 (Cbln1) is the prototype of a family of secreted neuronal glycoproteins (Cbln1-4) and its genetic elimination results in synaptic alterations in cerebellum (CB) and striatum. In CB, Cbln1 acts as a bi-functional ligand bridging pre-synaptic ß-neurexins on granule cells to post-synaptic Grid2 on Purkinje neurons. Although much is known concerning the action of Cbln1, little is known of the function of its other family members. Here, we show that Cbln1 and Cbln2 have similar binding activities to ß-neurexins and Grid2 and the targeted ectopic expression of Cbln2 to Purkinje cells in transgenic mice rescues the cerebellar deficits in Cbln1-null animals: suggesting that the two proteins have redundant function mediated by their common receptor binding properties. Cbln1 and Cbln2 are also co-expressed in the endolysosomal compartment of the thalamic neurons responsible for the synaptic alterations in striatum of Cbln1-null mice. Therefore, to determine whether the two family members have similar functions, we generated Cbln2-null mice. Cbln2-null mice do not show the synaptic alterations evident in striatum of Cbln1-null mice. Thus, Cbln2 can exhibit functional redundancy with Cbln1 in CB but it does not have the same properties as Cbln1 in thalamic neurons, implying one or both utilize different receptors/mechanisms in this brain region.