A glycoform of the secreted purple acid phosphatase AtPAP26 co‐purifies with a mannose‐binding lectin (AtGAL1) upregulated by phosphate‐starved Arabidopsis

Abstract

The purple acid phosphatase AtPAP26 plays a central role in Pi-scavenging by Pi-starved (-Pi) Arabidopsis. Mass spectrometry (MS) of AtPAP26-S1 and AtPAP26-S2 glycoforms secreted by -Pi suspension cells demonstrated that N-glycans at Asn and Asn were modified in AtPAP26-S2 to form high-mannose glycans. A 55 kDa protein that co-purified with AtPAP26-S2 was identified as a Galanthus nivalis agglutinin-related and apple domain lectin-1 (AtGAL1; At1g78850). MS revealed that AtGAL1 was bisphosphorylated at Tyr and Thr , and glycosylated at four conserved Asn residues. When AtGAL was incubated in the presence of a thiol-reducing reagent prior to immunoblotting, its cross-reactivity with anti-AtGAL1-IgG was markedly attenuated (consistent with three predicted disulfide bonds in AtGAL1's apple domain). Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating post-transcriptional control of AtGAL1 expression. Growth of a -Pi atgal1 mutant was unaffected, possibly due to compensation by AtGAL1's closest paralog, AtGAL2 (At1g78860). Nevertheless, AtGAL1's induction by numerous stresses combined with the broad distribution of AtGAL1-like lectins in diverse species implies an important function for AtGAL1 orthologs within the plant kingdom. We hypothesize that binding of AtPAP26-S2's high-mannose glycans by AtGAL1 enhances AtPAP26 function to facilitate Pi-scavenging by -Pi Arabidopsis.

Lectin AtGAL1 interacts with high‐mannose glycoform of the purple acid phosphatase AtPAP26 secreted by phosphate‐starved Arabidopsis

“…These observations are reminiscent of previous reports that although AtPAP26 activity and protein levels are substantially upregulated following Pi‐deprivation, AtPAP26 transcripts occur at similar levels in Arabidopsis suspension cells and seedlings irrespective of their nutritional Pi status (Hurley et al, ; Tran, Qian, et al, ; Veljanovski et al, ; Wang & Liu, ). Similarly, levels of intracellular and secreted AtGAL1 polypeptides showed a far greater increase during Pi‐deprivation of Arabidopsis relative to corresponding changes in AtGAL1 transcripts (Ghahremani et al, ).…”

“…AtPAP26‐S1, AtPAP26‐S2, AtPAP12, AtPAP25, and AtGAL1 were fully purified from cell wall extracts of −Pi Arabidopsis suspension‐cultured cells, and rabbit anti‐AtPAP26 immune serum (anti‐AtPAP26) and rat anti‐AtGAL1 immune serum (anti‐AtGAL1) obtained as previously described (Del Vecchio et al, ; Ghahremani et al, ; Robinson, Park, et al, ; Veljanovski et al, ). APase activity assays and protein concentration determinations were conducted as described by Ghahremani and co‐workers ().…”

“…These results led us to hypothesize that cotranslational and posttranslational glycosylation processes mediate functional diversity of secreted AtPAP26 glycoforms during Pi deprivation. Ghahremani and co‐workers () also documented extensive copurification of the high‐mannose AtPAP26‐S2 glycoform with a secreted, bisphosphorylated PSI lectin designated as AtGAL1 (At1g78850). AtGAL1 is a member of Arabidopsis ' Galanthus nivalis agglutinin (GNA) lectin family that bind high‐mannose glycans (Eggermont, Verstraeten, & Van Damme, ; Van Damme, Lannoo, & Peumans, ).…”

“…However, integrated biochemical and functional genomic studies have established the Arabidopsis PAP AtPAP26 (and its orthologue from rice, common bean, and stylo): (a) as a principal contributor to intracellular and extracellular PSI APase activity and (b) to play a central role in Pi‐scavenging and recycling during Pi deprivation or leaf senescence (Gao, Lu, Qiu, Wang, & Shou, ; Hurley et al, ; Liang, Sun, Yao, Liao, & Tian, ; Liu, Xue, Chen, Liu, & Tian, ; Robinson, Carson, Ying, Ellis, & Plaxton, ; Robinson, Park, et al, ; Shane, Stigter, Fedosejevs, & Plaxton, ; Tran et al, ; Veljanovski, Vanderbeld, Knowles, Snedden, & Plaxton, ; Wang & Liu, ; Wang et al, ). Native enzyme purification and characterization led to the discovery that AtPAP26 is secreted as a pair of distinct glycoforms (AtPAP26‐S1 and AtPAP26‐S2) by −Pi Arabidopsis (Del Vecchio et al, ; Ghahremani et al, ; Tran, Qian, et al, ). Our companion paper employed high‐resolution mass spectrometry to compare structural features of their N‐linked glycans (Ghahremani et al, ).…”

“…Native enzyme purification and characterization led to the discovery that AtPAP26 is secreted as a pair of distinct glycoforms (AtPAP26‐S1 and AtPAP26‐S2) by −Pi Arabidopsis (Del Vecchio et al, ; Ghahremani et al, ; Tran, Qian, et al, ). Our companion paper employed high‐resolution mass spectrometry to compare structural features of their N‐linked glycans (Ghahremani et al, ). Both AtPAP26 glycoforms shared identical glycosylation sites.…”