Cystatin C Elevated in Sanfilippo, Linked With Worse Lung Function
Levels of a protein called cystatin C are elevated in the lungs of people with Sanfilippo syndrome and related diseases, and higher levels of it are associated with worse respiratory function in these patients, a new study reports.
The findings also show that heparan sulfate, the molecule that accumulates to toxic levels in people with Sanfilippo, can inhibit cystatin C activity.
The study, “Binding of heparan sulfate to human cystatin C modulates inhibition of cathepsin L: Putative consequences in mucopolysaccharidosis,” was published in Carbohydrate Polymers.
Mucopolysaccharidoses, or MPS, are a group of genetic disorders marked by the toxic accumulation of molecules called glycosaminoglycans (GAGs). Sanfilippo syndrome, or MPS type 3, is mainly characterized by the accumulation of a GAG called heparan sulfate.
In prior work, the researchers — a team led by scientists in France — found that levels of cysteine cathepsins are reduced in respiratory secretions from people with Sanfilippo or other MPS types.
Cysteine cathepsins are a group of proteases, proteins responsible for cutting up other proteins into smaller pieces. The balance of proteases being “turned on and off” helps tissues to maintain their structural integrity. In the previous research, lower cysteine cathepsin levels were associated with worse respiratory function.
In this study, the team investigated a related protein called cystatin C, which normally inhibits the activity of cysteine cathepsins.
The scientists first investigated cystatin C levels in respiratory secretions from 11 MPS patients — four with Sanfilippo, two with MPS type 1, and five with MPS type 2. All of these MPS types are marked by the toxic accumulation of heparan sulfate. Secretions from nine people without MPS also were analyzed.
Results showed that cystatin C levels were significantly higher in the MPS patients, by about sevenfold on average. There was no apparent difference in cystatin C levels between MPS types.
Higher cystatin C levels were significantly correlated with worse respiratory function, as assessed with a measure the researchers had previously developed called global respiratory symptoms severity (GRSS). Higher heparan sulfate levels in the samples also were associated with worse respiratory function, in line with prior reports.
“This is the first study that evaluates the protein level of human cystatin C in respiratory specimen from MPS patients and correlates its expression with GRSS scores, which is associated to the severity of respiratory symptoms,” the researchers wrote.
Given that the MPS samples had high cystatin C levels, and that the protein blocks cysteine cathepsin activity, the researchers tested whether the MPS samples could inhibit the activity of a cathepsin protein in lab experiments. Unexpectedly, however, adding the MPS samples did not affect the activity of the protease.
One potential explanation for this unanticipated result could be that cystatin C is less effective in the presence of heparan sulfate and other GAGs. To test this hypothesis, the researchers pretreated the MPS samples with a cocktail of GAG-destroying enzymes and results showed these samples could powerfully inhibit the protease activity of cysteine cathepsins.
Further experiments using purified protein confirmed that the inhibitory effectiveness of cystatin C was markedly reduced in the presence of heparan sulfate.
“We identified HS [heparan sulfate] as an unexpected but potent modulator of the inhibitory activity of hCC [human cystatin C],” the researchers said. They speculated that this may alter the balance of protease activity in tissues in favor of excess protein destruction.
The scientists conducted a battery of lab experiments paired with molecular modeling to create a detailed model of the physical and chemical interactions between heparan sulfate and cystatin C.
“The structural analysis of the hCC/HS complex model presented here will facilitate further studies on the design of specific competitors aiming to prevent HS-hCC binding and ultimately restore the proteolytic cysteine cathepsin/cystatin balance,” they wrote.