Structure determination of the glycans of human‐serum α₁‐antichymotrypsin using ¹H‐NMR spectroscopy and deglycosylation by N‐glycanase

Abstract

a,-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into three microheterogeneous forms on a concanavalin-A -Sepharose column: a pass-through (peak I), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A -Sepharose column. The complete primary structure of the glycans was determined by high-resolution 'H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated ccl-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of u,-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alantichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak l contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.Human a,-antichymotrypsin is a plasma glycoprotein with a relative molecular mass ( M J of approximately 58000 and a carbohydrate content of 24% [l]. It belongs to the superfamily of serpins (serine proteinase inhibitors) [2]. In addition to Creactive protein and serum amyloid A protein, cr,-antichymotrypsin is one of the major acute-phase proteins [3] since its serum concentration increases more than fourfold within a few hours in response to an inflammatory stimulus [4].The precise biological function of a,-antichymotrypsin has not been yet determined. It is known to inhibit chymotrypsinlike proteases [5 -71 by formation of equimolar complexes which, however, dissociate with time. Eriksson et al.[8] recently described four families with inherited partial al-antichymotrypsin deficiency in which heterozygotes have 50% of normal plasma levels. No homozygotes have yet been identified. a,-Antichymotrypsin has been shown to be identical to the protein termed 64DP [9] and has a high affinity for DNAcellulose.It has been claimed that a,-antichymotrypsin is a component of the brain amyloid deposits in Alzheimer's disease [lo] but we recently showed 1111 that no distinction can be made between extracts of 14 control brains and extracts of 12 Alzheimer brains with regard to the amount of al-antichymotrypsin.The complete amino acid sequence of al-antichymotrypsin deduced from the nucleotide sequence [I21 reveals ...

Stimulatory effect of inflammatory cytokines on alpha 1-antichymotrypsin expression in human lung-derived epithelial cells.

“…These bands probably correspond to Achy with a number of Nglycan chains removed. This is assumed on the basis of previous studies where N-glycosidase F deglycosylation of serum Achy was performed (21). In accordance with this study five Achy forms were detected, indicating that serum Achy may carry four Asn-linked oligosaccharides.…”

Synthesis of α1-Antichymotrypsin and α1-Antitrypsin by Human Trophoblast

“…It is noteworthy that the a,-AT and a l -ACHY secreted by trophoblast have M, of 50 000 and 49 000, respectively, whereas the a,-AT and al-ACHY secreted by hepatocytes, monocytes, and epithelial cells and present in the circulation have M, of 54 000 and 68 000, respectively (I, 3, 5). The circulating forms of a,-AT and nl-ACHY are highly Nglycosylated (13% and 24% carbohydrate in three and four glycan chains, respectively) and the N-glycans are easily removed with PNGase F (19). Our finding that after deglycosylation the M, of the a,-AT and al-ACHY produced by trophoblast and HepG2 cells all appear to be the same suggests that the lower M, of the inhibitors secreted by the trophoblast results from a reduced level of glycosylation.…”

Human liver and human placenta both contain CMP‐NeuAc:Galβ1→4GlcNAc‐R α2→3‐ as well as α2→6‐sialyltransferase activity

“…[3H]glycoprotein and CMPNeuAc with human liver microsomes yielded a spot of 2,3, as the only trimethylated sugar (Fig:. 1, lane a).…”

“…By contrast, the tri-and tetra-antennary glycans of these glycoproteins have been shown to contain not only a2+6-but also cc2+3=linked sialic acid (references cited in [2,3]). Different methods have been employed to detect the human liver a3-sialyltransferase responsible for the synthesis of the a2+Zsialylated glycans.…”

“…Several studies from this and other laboratories have shown that the NeuAc linkage type generally can be explained from the activities of the CMP-NeuAc:Gal~1+4GlcNAc-R a2+3-(a3-sialyltransferase) or a2+6-sialyltransferase (cr6-sialyltransferase) in the corresponding tissues [ 15 I?]. However, no a3-sialyltransferase activity has been identified in human liver to date, although structural studies have revealed that the tri-and tetra-antennary glycans of several glycoproteins synthesized by this tissue do contain terminal NeuA~2-+3Gal/?l+4GlcNAc sequences (references cited in [2,3]). …”