Crystal Structure of Human Prostate-Specific Antigen in a Sandwich Antibody Complex

In Vitro Affinity Maturation of an Anti-PSA Antibody for Prostate Cancer Diagnostic Assay

“…Prompted by the structural analysis results of the Fab′ 5D3D11 -human PSA-Fab′ 5D5A5 ternary complex showing that none of the CDR-L3 residues establishes interactions with antigen (see Stura et al 12 ), we designed two structure-guided CDR-L3 libraries (named L3_PSA and L3 + 1_PSA). The L3_PSA library randomizes positions L92-L93-L94 using NNK codons and allows the selection of either Gly or Pro at position L95.…”

“…The difference between active PSA and aPSA lies in the distinct conformations of mature intact PSA. 11 This distinction can now be associated with the open and closed states of the catalytic cleft, wherein the activatable conformation is occupied by certain neighboring loops, excluding the kallikrein loop (see Stura et al 12 ).…”

“…However, a sandwich immunoassay using 5D3D11 as capture antibody and an anti-tPSA mAb as detection antibody yields a sensitivity that is not sufficient for good reliability. This is probably due to the modest binding affinity of 5D3D11 for PSA (dissociation constant K d ≈ 48 nM for Fab′ fragment; see Stura et al 12 ), prompting directed-evolution efforts to improve the affinity of 5D3D11 while maintaining its selective specificity. The principle of directed molecular evolution consists of cycles of diversification, selection, and amplification.…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…Apparently, the O-glycan rigidifies the surrounding surface loop region (Stura et al, 2011). Surprisingly, the corresponding site in KLK1 was not glycosylated, while not predictable O-glycosylation was observed in all Ser/ Thr residues of the N-glycosylated sequons (Kellermann et al, 1988).…”

“…Analyses of human urinary and seminal KLK3 samples corroborated varying N-glycosylation and O-glycosylation, which had a significant effect on the enzymatic activity against even small substrates, such as suc-Leu-Leu-Val-Tyr-AMC (Shibata et al, 1997). Whereas one KLK3 crystal structure (2ZCH, Figure 3) exhibited only two GlcNAc molecules linked to Asn61, another structure (3QUM) showed a triantennary N-glycan with terminal sialic acids and a mucin-type O-glycan (GlcNAc-Gal) at Thr125 (Menez et al, 2008;Stura et al, 2011). Similar to seminal KLK3, non-glycosylated KLK3 from E. coli expression reacts with anti-chymotrypsin.…”