Role of hyaluronan and proteoglycan link protein

Link protein was discovered as one of the major components of the extracellular matrix (ECM) of abundant cartilage tissue. Biochemical analysis and electron microscopic observation of ECM components isolated from cartilage tissue revealed that a single Link protein molecule binds to a single aggrecan (ACAN) molecule, the major proteoglycan (PG) of cartilage tissue, synergistically stabilizing those non-covalent binding to hyaluronan. The molecular structure of link protein is very similar to the N-terminal G1 domain of the hyaluronan-binding aggrecan family (or lectican family, since it contains a C-type lectin motif in its G3 domain). The B, B' subdomains are approximately 100 amino acid residues long and are also called proteoglycan tandem repeat (PTR) or link modules and are abundant in the link module superfamily with hyaluronan-binding properties. Until the discovery of other family members by molecular cloning techniques, the link protein abundant in cartilage tissue was called cartilage link protein (CRTL1). Three other family members have been cloned after 2000 and they were named the hyaluronan and proteoglycan link protein (HAPLN) gene family (1). Interestingly, all four HAPLN genes are present in the genome in pairs with lectican PG genes such as ACAN. The link module superfamily of hyaluronan-binding genes is a hallmark of vertebrates, and the four paired genes were probably created during evolution.

HAPLN1-deficient mice showed defects in cartilage development and delayed bone formation with short limbs, and craniofacial abnormalities (2). And most mice died of respiratory failure shortly after birth. Although the chondrodysplastic symptoms were milder than in spontaneous Acan mutant (cmd/cmd) mice, ACAN deposition was reduced to about 25% and the columnar structure of growth plate chondrocytes was disorganized. These results suggest that HAPLN1 is important for the formation of PG aggregates, chondrocyte differentiation and normal organization (2). In addition, VCAN expression was decreased during cardiac development in HAPLN1-deficient mice and abnormalities such as ventricular septal defects were observed, suggesting that VCAN-Hyaluronan-HAPLN1 aggregate formation is important for cardiac development.

Among the HAPLN gene family, Hapln2 and Hapln4 are expressed specifically in the central nervous system, and Hapln1 is also found to be expressed in the brain and cardiovascular system in addition to cartilage; Hapln3 expression is ubiquitous (1). It is likely that individual HAPLNs are expressed in a tissue- and time-specific manner and function by regulating the stabilization of lectican PG binding to hyaluronan.

In brain development, HAPLN1 expression peaks from embryonic to juvenile stages, while HAPLN2 and HAPLN4 expression increases toward maturity. The temporal pattern of the former is more consistent with neurocan (NCAN) and VCAN V0/V1 (V indicates splicing variant), while the latter is more consistent with ACAN, brevican (BCAN), and VCAN V2 (Fig. 1). In addition to differences in temporal expression, whether these ECM components are expressed from neurons/glial cells is related to the formation of site-specific hyaluronan-binding PG aggregates in the brain. In this context, the initiation of HAPLN expression is a trigger for aggregate formation. Another difference from ACAN aggregates in cartilage tissue is the cross-linking of lectican G3 domains by tenascin in brain. It has been named the HLT model, an acronym for hyaluronan-lectican-tenascin (3).

Fig.1. Expression of HAPLN and chondroitin sulfate proteoglycan (CSPG) genes during brain development. Abbreviations: TN-C, tenascin C; TN-R, tenascin R

The perineuronal net (PNN) structure discovered by Golgi and Ramon y Cajar et al. is a mesh-like structure found in the cell bodies and dendrites of certain neurons but is a highly condensed ECM structure on the cell membrane surface. Since HAPLN1 systemically deficient mice are lethal early in life due to chondrodysplasia, analysis of PNN development after birth in mice with rescued expression in cartilage (Crtl1^-/-/Crtl1-Tg^+/+) showed that PNN accumulation in the cortex was greatly attenuated without significantly affecting the overall expression level of PG (4). Furthermore, the same mice maintained ocular dominance plasticity in the adult. These results indicate that HAPLN1 plays a critical role in the formation of PNNs and the regulation of their plasticity. In contrast, HAPLN4 is expressed predominantly in the cerebellum and brainstem nuclei, where, however, HAPLN1 also co-localizes; in these nuclei of HAPLN4-deficient mice, it affects the localization pattern of the BCAN but not the ACAN. In the deep cerebellar nuclei, our results elucidate HAPLN4 as a PNN component that is selectively responsible for GABAergic Purkinje cells- deep cerebellar nuclei synapse formation (5).

HAPLN2 expression is exclusively in the white matter of the brain and is prominently localized to the nucleus of Ranvier (NOR). The NORs found in myelinated nerves are located between the myelin sheath covering the nerve axon, where voltage-gated sodium channels cluster and action potentials are generated. We found that the localization of HAPLN2 in CNS NORs is colocalized with that of VCAN V2, followed by the localization of BCAN and NCAN in many but not all CNS NORs. We showed that BCAN localizes only to NOR, which has a large axonal diameter, and that tenascin-R and phosphacan colocalize with it, based on immunostaining and immunoprecipitation experiments using the optic nerve from BCAN-deficient mice (6). The high affinity of the fibronectin type III repeat of tenascin-R to the G3 domain of BCAN could explain this phenomenon. The larger axon diameter NORs contain more complex ECM aggregates, including strongly negatively charged carbohydrate chains such as chondroitin sulfate chains, which may be related to the higher saltatory conduction velocity of the larger myelinated nerves (6).

In the brain, HAPLN plays a central role in the formation of perineuronal ECMs, which have been known for a long time and have recently attracted attention for their functions in controlling neuroplasticity, psychiatric disorders, and memory. The above two types of perineuronal ECMs are thought to create a “field” where various signals intersect (7).

Toshitaka Oohashi
(Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University)

Jun 15, 2023