Glycan-based biomarkers for mucopolysaccharidoses

Abstract

The mucopolysaccharidoses (MPS) result from attenuation or loss of enzyme activities required for lysosomal degradation of the glycosaminoglycans, hyaluronan, heparan sulfate, chondroitin/dermatan sulfate, and keratan sulfate. This review provides a summary of glycan biomarkers that have been used to characterize animal models of MPS, for diagnosis of patients, and for monitoring therapy based on hematopoietic stem cell transplantation and enzyme replacement therapy. Recent advances have focused on the non-reducing terminus of the glycosaminoglycans that accumulate as biomarkers, using a combination of enzymatic digestion with bacterial enzymes followed by quantitative liquid chromatography/mass spectrometry. These new methods provide a simple, rapid diagnostic strategy that can be applied to samples of urine, blood, cerebrospinal fluid, cultured cells and dried blood spots from newborn infants. Analysis of the non-reducing end glycans provides a method for monitoring enzyme replacement and substrate reduction therapies and serves as a discovery tool for uncovering novel biomarkers and new forms of mucopolysaccharidoses.

Keywords: Carbohydrate biomarkers; Glycosaminoglycans; Lysosomal storage disorders; Mass spectrometry; Mucopolysaccharidoses; Sensi-Pro assay.

Fig. 1

Glycosaminoglycan catabolism. The schemes show the different enzymatic activities required for the sequential catabolism of a hypothetical NREs from heparan sulfate, dermatan sulfate and keratan sulfate. It should be noted that the glucuronic acid 2-O-sulfatase in heparan sulfate degradation has been demonstrated in vitro, but has not yet been identified genetically. Scheme modified from [3] according to findings from Lawrence et al. [18] and Kowalewski et al. [87].

Fig. 2

Scheme for determining NREs and internal disaccharides. Enzymatic depolymerization of HS with heparan lyases releases internal disaccharides (dashed arrows) that contain an unsaturated uronic acid. The NRE liberated from the left end of the chain as drawn lacks the Δ^4,5-double bond and is 18 Da greater in mass than a corresponding internal disaccharide. Reductive amination with [¹²C₆]aniline facilitates separation of the various disaccharides by LC/MS and gives the indicated m/z values for the molecular ions shown in parentheses. The glycan structures are graphically represented by geometric symbols, which are defined in the lower part of the figure [88]. To simplify the representation of constituent disaccharides, we use a structure code (DSC) [89]. In the DSC, a uronic acid is designated as U, G, I or D for an unspecified hexuronic acid, d-glucuronic acid, l-iduronic acid or Δ^4,5-unsaturated uronic acid, respectively. The hexosamines are designated in upper case for glucosamine and lower case for galactosamine, and the N substituent is either H, A,S or R for hydrogen, acetate, sulfate or some other substituent, respectively. The presence and location of ester-linked sulfate groups are depicted by the number of the carbon atom on which the sulfate group is located or by 0 if absent. For example, I2S6 refers to a disaccharide composed of 2-sulfoiduronic acid-N-sulfoglucosamine-6-sulfate, whereas D2S6 refers to a similarly structured disaccharide that instead has a Δ^4,5-double bond in the uronic acid. Adapted from [18] with permission.

Fig. 3

Systematic diagnostic screening of GAG samples for various MPS disorders. (a) As an example, the process of uncovering the NRE biomarkers in MPS I and MPS IIIA samples is shown. Dashed circles indicate specific NRE structures for these two disorders. (b) The flow chart illustrates how MPS diagnosis can be carried out. The detection criteria are tied to the exact structure of the biomarkers based on size (monosaccharide, disaccharide or trisaccharide) and structural features such as the number of acetates (Ac) and sulfates (S). For a complete unknown, NRE analysis would be carried out on both HS and CS/DS as indicated. The key to the symbols is shown in Fig. 2.

Fig. 4

Newborn screening of various MPS disorders. A portion of a newborn bloodspot sample was immersed in a solution of Pronase and incubated overnight to solubilize GAGs. The GAGs were then purified by anion exchange chromatography using DEAE-Sepharose, washing away contaminants with 0.2 M NaCl. The GAGs were eluted with1 M NaCl. After desalting, the GAG chains were enzymatically depolymerized with heparan lyases and the released component residues including both NRE and internal unsaturated disaccharides were derivatized with [¹²C₆]aniline. The derivatized samples were mixed with standards NRE biomarkers labeled with [¹³C₆]aniline to distinguish them from the NRE residues present in the biological samples. The mixed samples were next analyzed by LC/MS and based on the biomarker profile a tentative diagnosis was determined using the logic mapped out in the decision tree shown in Fig. 3.