N-glycans of human protein C inhibitor: tissue-specific expression and function

Abstract

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH₂-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc₃Hex₅HexNAc₄, consistent with a core fucosylated bi-antennary glycan with terminal Lewis(x). A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH₂-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M⁻¹ s⁻¹ for the natural full-length PCI and a form lacking six amino acids at the NH₂-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M⁻¹ s⁻¹ for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH₂-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.

Figure 1. Western blot and SDS-PAGE image of purified PCI.

Human seminal and blood plasma PCI were purified as described in Materials and Methods. Products were analyzed by SDS-PAGE, followed by staining with Coomassie Brilliant Blue R-250 or subjected to western blot. Lane 1, western blot analysis of blood plasma PCI; Lane 2, western blot analysis of seminal plasma PCI; Lane 3, SDS-PAGE of seminal plasma PCI.

Figure 2. MALDI-TOF mass spectrum of permethylated PCI N-glycans from human seminal plasma.

N-glycans were derived from 50% (v/v) acetonitrile fraction from a C₁₈ Sep-Pak. All molecular ions are [M+Na]⁺, and nomial masses of the ¹²C isotope are shown. Putative structures based on composition and knowledge of biosynthetic pathways are shown. The symbols for the monosaccharide units are explained in the picture.

Figure 3. Native PAGE of desialylated blood plasma PCI.

PCI was desialylated,,purified and analyzed by native PAGE, as described in Materials and Methods. The gel was stained with Ruby protein stain. Lane 1, native PCI; Lane 2, desialylated N-terminally cleaved PCI; Lane 3, desialylated full-length PCI.

Figure 4. Rate of PSA inhibition by full-length native PCI derived from blood.

The inhibition of PSA was measured as a function of time by a discontinuous assay, as described in Materials and Methods.

Figure 5. Effects of N-glycans and the Δ6-N-terminus on the rate of PCI inhibition of PSA.

The inhibition of PSA by untreated full-length, untreated Δ6-N-cleaved, deglycosylated full-length, deglycosylated Δ6-N-cleaved, desialylated full-length and desialylated Δ6-N-cleaved PCI isolated from blood plasma were measured as a function of time by a discontinuous assay. The resulting second-order rate constants are displayed. Data are the mean ± SD of two independent experiments, each based on 4–6 time points.

Figure 6. Modeling of the NH₂-terminal parts of PCI and the PCI-PSA complex.

(A) Ribbon cartoon of PCI (PDB entry 2OL2) with the fantasy model of the NH₂-terminal part. Residues 1–6 (HRHHPR), 7–10 (EMKK) and 11–28 (R11 – D28) of the fantasy model are shown in cyan, orange and black respectively. N-glycan-attaching residues and the RCL loop, which interact with PSA in the PCI-PSA complex, are shown in blue and brick red, respectively. Amino- and carboxy-termini are labeled NH2 and COOH, respectively, where the surface of PCI is shown in light grey. (B) Stereo illustration of the surface of PSA, displaying the catalytic pocket forming loops in orange. The catalytic residues are marked in red. The RCL loop of PCI binds to this catalytic pocket.

Figure 7. Tissue-specific expression of N-glycans on PCI.

Major N-glycans of PCI derived from blood plasma, seminal plasma and urine are framed in light red, blue and yellow, respectively. N-glycan structure of urinary PCI is drawn according to . * represents the most abundant glycan. Glycan marked with “S” has a special lacdiNAc (GalNAcβ1-4GlcNAc) structure (constitutes about 12% of the total N-glycans in urinary PCI [13]). This structure has not been observed in blood or seminal plasma PCI N-glycans.