Laminin β2 was identified as basement membrane a component in neuromuscular junctions (NMJs). Basement membranes are thin sheet-like structures that abut many cell types, including epithelial, endothelial, fat, muscle, and peripheral nerve cells. They influence tissue compartmentalization and cellular phenotypes during developmental and regenerative processes. Although a pure basement membrane was necessary to investigate its composition, it was difficult to purify the basement membrane from various tissues, including NMJs. The Matrigel matrix, an extract derived from mouse Engelbreth-Holm-Swarm sarcoma, is composed of type IV collagen, laminin, nidogen, and perlecan, which are the major components of basement membranes. Therefore, the Matrigel matrix has been used as a reconstituted basement membrane. Although Matrigel has significantly advanced many studies of basement membranes, several reports have suggested that the basement membrane in NMJs is more specific than that in other tissues. To characterize the molecules of the synaptic basement membrane, Sanes et al. adopted an immunohistochemical approach (Sanes et al. 1990). A specific molecule, laminin β2, was initially defined by an antiserum to an alkali digest of bovine lens capsule that is a portion of the basement membrane. The antiserum, named JS-1, recognized synaptic basement membrane rather than the extrasynaptic membrane in adult rat muscle (Sanes and Hall 1979). Additional monoclonal antibodies, C4, D5, and D7, were generated against the same antigen. The cDNAs encoding the recombinant proteins reactive to these monoclonal antibodies were isolated from a rat kidney-derived library. Analysis of these cDNA sequences revealed homologies with laminin subunits. Therefore, the specific antigen in NMJs was initially named s-laminin. After the first revision of laminin nomenclature (Burgeson et al. 1994), s-laminin was renamed laminin β2, and the gene was named LAMB2.
Structure and Function of Laminin β2
Laminins are major cell-adhesive proteins that are capable of binding to integrins. Integrins are heterodimeric membrane proteins that are composed of noncovalently associated α and β subunits. To date, 24 integrins have been identified in mammals (Humphries et al. 2006). Of these integrins, integrins α3β1, α6β1, α6β4, and α7β1 have been identified as the major laminin receptors in various cell types (Durbeej, 2010). The α7X1 and α7X2 subunits, which contain alternatively spliced X1 or X2 regions in the extracellular β-propeller domain, add diversity to the α7β1 integrin. Of integrins binding to laminins, integrin α3β1 is classified as an X2-type integrin. The α3β1 and α7X2β1 integrins are capable of distinguishing between β1- and β2-containing laminins (Taniguchi et al. 2009). The C-terminal 20 amino acid sequence of the laminin β2 chain modulates the binding affinities of laminins to X2-type integrins. Because β2-containing laminins and X2-type integrins colocalize in several tissues, the high affinity interactions between them may contribute to the functions that are not compensated by the laminin β1 chain.
Expression and Distribution of Laminin β2
Initially, laminin β2 was considered to have a restricted distribution pattern based on the selective staining of NMJs in skeletal muscle and glomerulus in kidney by specific anti-laminin β2 antibodies (Hunter et al. 1989b). Thereafter, several groups revealed that the β2 chain dominates at sites such as the synaptic cleft of NMJs, the glomerular basement membrane (GBM), and the basement membrane of the retina, although β1 and β2 chains are ubiquitously expressed in various tissues (Hunter et al. 1992; Iivanainen et al. 1995; Miner et al. 1997; Patton et al. 1997; Sasaki et al. 2002). The distribution patterns of β2-containing laminins are further restricted by the expression of α and γ chains (Fig. 2) (Durbeej 2010).
In adult peripheral nerves, myelinated axons and fascicles of myelinated axons are surrounded by endoneurial and perineural basement membranes, respectively. LM-421 and LM-521, which contains the β2 chain, are present in perineural basement membranes, and endoneurial basement membranes contain LM-211(Patton et al. 1998). The endoneurial basement membrane that is close to NMJs contains LM-411. As described above, laminin β2 is concentrated in the synaptic basement membrane. An in vitro study showed that the C-terminus of the β2 chain is necessary for the synaptic localization of laminin β2 (Martin et al. 1995).
Consistent with the distribution pattern of laminin β2 in NMJs of skeletal muscles and in GBMs of kidney, mice lacking laminin β2 (Lamb2−/−) exhibit developmental defects in neuromuscular synapse formation and glomerular filtration (Noakes et al. 1995a; Noakes et al. 1995b). Therefore, Lamb2−/− mice die between postnatal days 15 and 30. The mutant mice also exhibit defective rod photoreceptor synapses due to the aberrant elongation of the outer segment (Libby et al. 1999). Genetic deletion of laminin β2 and γ3 disrupts the development of the basement membrane of the retina, i.e., the inner limiting membrane, which maintains the apical-basal polarity of Müller cells, astrocyte migration, and vascular integrity (Gnanaguru et al. 2013; Pinzon-Duarte et al. 2010). Thus, deletion of laminin β2 causes defects in NMJs, kidney, and retina.
Roles of Laminin β2 in NMJs
Synaptic transmission at NMJs directs the contraction or relaxation of skeletal muscle. An NMJ is a chemical synapse that is formed between a motor neuron and a muscle fiber (Fig. 3a). At NMJs, an axon terminal occupies a depression of the muscle fiber and forms a synaptic cleft with junctional folds, which are filled with basement membrane containing laminin β2. The axon terminal contains mitochondria and synaptic vesicles filled with the neurotransmitter acetylcholine. The neurotransmitter is released at active zones, which are protein-dense areas of the presynaptic plasma membrane. The neurotransmitter release is initiated by the calcium ion influx through voltage-gated calcium channels (VGCCs). The released acetylcholine binds to acetylcholine receptors (AChRs) at crests of the junctional folds of the muscle plasma membrane to accomplish synaptic transmission.
The roles of laminin β2 in NMJs are exemplified in the defective NMJs of Lamb2−/− mice (Noakes et al. 1995a). In the Lamb2 mutant mice, active zones disappear and synaptic vesicles are not concentrated at nerve terminals that abut muscle fibers. In search for laminin β2 specific receptors, β2 chain of LM-421 and LM-521 were identified as ligands that bind to the eleventh extracellular domain of N- and P/Q-type VGCCs (Fig. 3b) (Nishimune et al. 2004). These VGCCs are concentrated in presynaptic terminals at NMJs and are required for neurotransmitter release from motor nerve terminals. However, LM-521 does not bind to R- and L-type VGCCs, which are not concentrated at NMJs. Interestingly, laminin β2-containing synaptic laminins bind to P/Q-type VGCCs, but β1 chain-containing nonsynaptic laminins (LM-111) do not bind to any of these VGCCs. These results suggest that synaptic laminins containing laminin β2 bind specifically to VGCCs concentrated at NMJ presynaptic terminals. In addition, P/Q-type VGCCs distinguish laminins containing laminin β2 from those without laminin β2. Importantly, the number of active zones decreases significantly when the interaction between laminin β2 and P/Q-type VGCCs is inhibited acutely in wild-type mice in vivo (Nishimune et al. 2004). Consistently, double knockout mice lacking N- and P/Q-type VGCCs exhibit a decreased number of active zones in otherwise normally formed NMJs (Chen et al. 2011a). The active zone loss phenotype identified in these in vivo experiments does not seem to be a secondary phenotype to NMJ degeneration. These results suggest that the interaction between synaptic laminins with laminin β2 and presynaptic VGCCs organizes active zones at NMJs.
Hunter et al. showed that chick ciliary neurons (parasympathetic neurons innervating ocular muscles) adhere to a preparation containing laminin β2 chain (Hunter et al. 1989b). They further identified a primary sequence in the C-terminus of the β2 chain that is involved in the adhesion of motor neurons (Hunter et al. 1989a). Recombinant proteins and synthetic peptides containing the identified LRE (Leucine-Arginine-Glutamate) sequence exhibit adhesive activities for ciliary neurons (Hunter et al. 1991). The LRE-containing C-terminal 20 kDa region of laminin β2 is also necessary and sufficient for laminin interactions with VGCCs (Nishimune et al. 2004). The VGCC is a candidate receptor that mediates the adhesion of motor neurons to laminin β2. The 100 amino acid sequence surrounding the LRE sequence is very well conserved among human, mice, and rats; however, the LRE motif is not conserved in the human β2 chain. The human laminin β2 chain contains a tripeptide LRG (Leucine-Arginine-Glycine) sequence in the corresponding region. It has not been tested whether the LRG sequence has adhesive activity for neurons and/or binds to VGCCs.
In Lamb2−/− mice, Schwann cells intrude into the synaptic cleft, the junctional folds are lost, and nerve terminals detach from muscle fibers (Noakes et al. 1995a). An in vitro study showed that LM-521, but not LM-221, inhibits process extension of Schwann cells (Patton et al. 1998). The LRE sequence of laminin β2 is not necessary for the Schwann cell repellent effect. These results suggest that Schwann cells use a receptor different from the neuronal receptors recognizing the LRE sequence in laminin β2. Furthermore, this receptor is likely to recognize a combination of laminin α5, β2, and γ1 chains, not just the β2 chain, as VGCCs do. The receptor that repels Schwann cells from the NMJ synaptic cleft has not yet been identified.
In addition to the active zone and Schwann cell phenotypes, Lamb2−/− mice exhibit neurotransmission impairment at NMJs (Knight et al. 2003; Noakes et al. 1995a). The miniature endplate potential frequency and the mean quantal content are both significantly decreased in Lamb2−/− NMJs compared to control NMJs. Interestingly, Lamb2 mutant NMJs also exhibit a failure to switch from N- and P/Q-type VGCC-mediated synaptic transmission during development to P/Q-type VGCC dependent transmission at mature NMJs (Chand et al. 2015). These physiological phenotypes are consistent with the aforementioned active zone loss phenotype that was identified in Lamb2−/− mice because a loss of active zones is equal to a loss of synaptic vesicle release sites. Furthermore, these symptoms in Lamb2−/− mice are consistent with the symptoms of patients with Pierson syndrome with Lamb2 gene mutations who show neuromuscular defects, renal failure, and severe ocular abnormalities (Matejas et al. 2010).
Roles of Laminin β2 in Glomerulus
Laminins have been used as culture substrata for stem cells. Long-term self-renewal of human pluripotent stem cells in defined culture systems needs to be achieved without mouse feeder cells. LM-521, but not LM-511, enables clonal culturing of human embryonic stem cells under defined and xeno-free conditions (Rodin et al. 2014). However, the advantage reason of the β2 chain remains unclear.
The development of biomimetic materials is a focus in the field of tissue engineering. Biologically active sequences derived from laminins and other extracellular matrices have been used to design biomimetic materials (Rahmany and Van Dyke 2013). The LRE sequence of the β2 chain may be a useful material for modulating the direction of neurite extension.
Laminin β2 was originally identified as a specific antigen in NMJs. It is a subunit of laminin chains and assembles with α and γ chains. Laminin heterotrimers are a family of glycoproteins that are major components of the basement membrane, in which they play structural and functional roles. Laminin β2 has unique expression patterns and restricted localization in basement membranes. It is preferentially found in the NMJ synaptic cleft in skeletal muscles, the GBM of kidney, and the inner limiting membrane of the retina. Consistent with the distribution pattern, mice lacking laminin β2 exhibit defects in neuromuscular and retinal synapse development and glomerular filtration. The defects of Lamb2−/− mice correspond to the clinical manifestations of Pierson syndrome, which is caused by mutations of the LAMB2 gene that encodes laminin β2. Forced expression of laminin β2 with Pierson syndrome mutations in Lamb2−/− mice ameliorates the severe nephrosis and neuromuscular abnormalities identified in Lamb2−/− mice. Upregulation of laminin β2 in GBM and NMJs is a potential therapeutic target in Pierson syndrome.
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