The proteoglycan bikunin has a defined sequence

Abstract

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan, with a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike nucleic acids and proteins, whose synthesis is template driven, Golgi-synthesized glycosaminoglycans are not believed to have predictable or deterministic sequences. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size-similar and charge-similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular ions corresponding to monocompositional peptidoglycosaminoglycans. Fragmentation using collision-induced dissociation unexpectedly afforded a single sequence for each monocompositional parent ion, unequivocally demonstrating the presence of a defined sequence. The biosynthetic pathway common to all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment in the understanding of information storage in complex glycans.

Figure 1

Biosynthetic pathway for chondroitin sulfate A GAG, on a serine (Ser) residue of the core protein, beginning in the endoplasmic reticulum and concluding in the Golgi apparatus. The biosynthetic enzymes are: β1XylT-I, β-xylosyl transferase I; β4GalT-I, β-4-galactosyl transferase I; β3GalT-II, β-3-galactosyl transferase II; β3GlcAT-I, β-3-glucuronosyl transferase I; β4GalNAcT-I, β-4-N-acetyl galactosaminyl transferase I; β3GlcAT, β-3-glucuronosyl transferase; β4GalNAcT, β-4-N-acetyl galactosaminyl transferase; ChSy, chondroitin synthases; and C4STs, galactosyl 4-O-sulfo transferase and N-acetyl galactosaminyl-4-O-sulfotransferase.

Figure 2

FT-ICR-MS analysis of polydisperse and heterogeneous bikunin pG. “Quad-windowed” mass spectra are depicted with individual narrow m/z acquisitions in unique colors.

Figure 3

FT-ICR-MS analysis of a bikunin pG fraction. a. FT-ICR negative-ion mass spectrum of 5.80 kDa M_R fraction by PAGE with 18 isobars and 63 parent-ions; b. Deconvolution of spectrum a; c. CID-FT-ICR-MS/MS spectra of parent-ion m/z = 917.38 (z = 6) and annotated fragment-ions providing sequence with dp27-5-Ser fragmentation pattern assigned from spectrum.

Figure 4

Bikunin sequencing flow chart (From left to right). The M_R (kDa) determined based on PAGE of fractions (blue rectangles represent gel bands) of bikunin in pG prepared by continuous elution PAGE is shown. The deconvoluted MS obtained using FT-ICR-MS affords the mass of 3-5 odd and even components (green ovals) observed in each bikunin pG fraction is shown. Each MS spectrum showed multiple change states (z-values) shown as red diamonds from which parent-ions were selected for MS/MS giving CID fragments by analysis on FT (purple circles) or FT-ICR (brown circles). The composition is designated dp and sulfo group number (i.e., dp 27-5-Ser is 27 saccharide units with 5-sulfo groups O-glycosidically linked to a Ser residue). A shorthand sequence for each chain is shown with a, b, c and d subdomain repeats indicated by numbers (i.e., 0.2.0.3 for d = 0, c = 2, b = 0, a = 3). The overall sequence of bikunin CS-A pG shown at the bottom is consistent with all determined sequences.