Brissett, Nigel Craig; (1997) Structural studies of domains in aggrecan and link protein from human cartilage. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Aggrecan and link protein are major components of cartilage. They play a key role in the maintenance of tensile and elastic properties of cartilage. In order to understand this role further, a structural study of the globular domains in aggrecan and link protein was carried out. A major function of aggrecan and link protein is to bind to hyaluronic acid (HA) which they do via proteglycan tandem repeats (PTRs). The structure of the PTR fold was uncharacterised. Thus, consensus secondary structure predictions were carried out for 59 PTR sequences and compared with predictions from 129 C-type lectin sequences. It emerged that both sets of sequences possess similar secondary structure patterns. Averaged protein fold recognition analyses showed that the PTR sequences are highly compatible with the C-type lectin fold. Subsequent molecular modelling of the PTR fold suggested that conserved basic residues are ideally placed for HA binding. As the G3 region of aggrecan also possesses a C-type lectin fold, structural studies were carried out on this region. In turn, consensus secondary structure predictions and averaged fold recogniton analyses for 131 carbohydrate recognition domain (CRD) sequences were carried out. The 13 G3 CRD sequences correlated with these predictions and the CRD protein fold as well. Alignment of 13 G3 short consensus repeat (SCR) sequences showed agreement with the a consensus analysis of 101 SCR sequences. The CRD and SCR domains were joined to create a model for 03 in human aggrecan. Electrostatic potentials for this model showed an extended region of positive charge at the interdomain cleft between the CRD and SCR domains. The existence of this cleft is compatible with binding to glycosaminoglycans. In addition to the modelling data, the G3 region was expressed in E. coli for structural and functional work. Purification and characterisation of the expressed products was achieved.

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