Reduced Heparan Sulfate Accumulation in Enterocytes Contributes to Protein-Losing Enteropathy in a Congenital Disorder of Glycosylation

Abstract

Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided. Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an ␣1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide. The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported. The mutations were tested for their ability to rescue faulty N-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain. Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective. Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast. Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress. 1-4 These defects reduce the amount or change the structure of the lipid-linked oligosaccharide (LLO) precursor for N-linked glycosylation, leading to underglycosylation of many proteins, 3 including antithrombin-III, factor XI, or protein C. This reduces their levels, leaving patients at risk for coagulopathy. Mutations in phosphomannose isomerase (MPI, causing CDG-Ib) and phosphomannomutase (PMM2, causing CDG-Ia) reduce the amount of GDP-Man, the immediate precursor of LLO,5 thus reducing the amount of N-linked glycosylation. Mutations in ALG6, which encodes an ␣1,3-glucosyltransferase, cause CDG-Ic. 6 -8 This enzyme is required for the addition of the first of three glucose residues to LLO, and without the first glucose, further glucosylation is prevented. The nonglucosylated precursor oligosaccharide is a poor substrate for the oligosaccharyltransferase complex and is inefficiently transferred to proteins. 9,10

ALG6-CDG in South Africa: Genotype-Phenotype Description of Five Novel Patients

“…The p.A333V mutation is associated with a mild to moderate phenotype which indicates that the novel frameshift mutation (c.1338dupA), which results in the formation of a truncated ALG6 protein, probably explains the severe phenotype and early death of the patient as well as the unusually low serum tetrasialotransferrin. The phenotypic variation from the typical ALG6 deficiency concurs with previous literature of rare ALG6-CDG cases with multiple organ involvement and intestinal abnormality, e.g., PLE (Westphal et al 2000b;Newell et al 2003;Al-Owain et al 2010). Patients 2, 3, 4, and 5 presented with the classical ALG6-CDG phenotype comprising of a variable degree of neurological involvement, including hypotonia and resistant epilepsy.…”

“…Patient 1 showed a severe, earlyonset, neuro-gastrointestinal presentation comparable with previously described case studies (Westphal et al 2000b;Damen et al 2004;Newell et al 2003). The patient was compound heterozygous for the prevalent c.998C>T (p.A333V) missense mutation (originally described by Imbach et al 1999) and a novel c.1338dupA (p.V447SfsX44) frameshift mutation in the ALG6 gene.…”

Heparan Sulfate Plays a Central Role in a Dynamic in Vitro Model of Protein-losing Enteropathy

“…In protein-losing enteropathy (PLE), 2 where plasma proteins leak into the intestine, this essential barrier is impaired (1)(2)(3)(4). PLE develops as a life-threatening complication of seemingly unrelated diseases, e.g.…”

Identification of a frequent variant inALG6, the cause of Congenital Disorder of Glycosylation-Ic

“…The Saccharomyces cerevisiae strain YG227 (Reiss et al, 1996) deficient in ALG6 (a kind gift from Markus Aebi) was grown in standard YPD and SC media (Sherman, 1991). The α1,3 glucosyltransferase complementation using patients ALG6 was done as described (Westphal et al, 2000a;Westphal et al, 2002).…”

“…We previously identified a CDG-Ic patient with three mutations in ALG6, one maternal mutation, causing p.Ala333Val, and two paternal mutations, causing p.Ser308Arg and p.Tyr131His (Westphal et al, 2000a). Each mutation was tested for its ability to complement the defective glycosylation of carboxypeptidase Y (CPY) in an alg6 deficient-strain of S. cerevisiae.…”