# The Heat Shock Protein 60 and Pap1 Participate in the _Sporothrix schenckii_-Host Interaction
## 3 Results  
### Polypeptide Composition of Peptidorhammonium  
Although the PRM carbohydrate composition was studied and the structure of the main glycans was already solved [19; 20; 21; 23; 24], little is known about the peptide backbone of this cell wall complex. PRM was extracted from the yeast-like cells wall, following the conventional methodology described in Material and Methods section. When this fraction was analyzed by denaturing SDS-PAGE, no defined protein bands were identified (Figure 1A), most likely due to the high degree of protein glycosylation. When the PRM was deglycosylated by treatment with \(\beta\)-elimination and endoglycosidase H, most of the dispersed material found in the PRM electrophoretic mobility disappeared and concentrated to the bottom of the lane and two faint protein bands of 65 kDa and 54 kDa were observed (Figure 1A). To confirm the PRM glycosylation, the sample was analyzed by lectin blot, using ConA-FITC, and under these conditions, protein bands with molecular weights in the rank of 90-55 kDa were identified, and a 75 kDa protein band being the most abundant in the PRM preparation (Figure 1B). When the lectin blot was repeated with the deglycosylated material, most of the signals observed in the glycosylated samples were lost, and the labeling of the 75 kDa protein was weaker in the deglycosylated PRM (Figure 1B). The deglycosylated PRM was subjected to capLC-ESI-QTOF/MS and MS/MS, yielding 325 polypeptides (Supplementary Material, Table S1), and from these, 26 main proteins were identified with Andromeda scores above 100 and with \(q\) values equal to zero [48] (Table 1). From this table, we decided to work with the chaperonin GroEL-like protein, also known as heat shock protein 60 (Hsp60), because it was reported to be involved in the pathogen-host interaction in other fungal species and we obtained the highest sequence coverage (48.9%) and Andromeda score (323) for this protein. The protein was isolated from both prokaryotic and eukaryotic cells, and in the latter, the canonical function is performed in the mitochondria, assisting the folding of proteins with regions of exposed hydrophobic \(\beta\)-sheets and molecular weight lower than 60 kDa [65]. However, it was also reported as a moonlighting protein found in the cell wall of _H. capsulatus_ and _P. brasiliensis_. Regarding the former, it was reported as a highly immunogenic glycoprotein that participates in adhesion to the host, and vaccination with a recombinant version protects mice from the deep-seated histoplasmosis [36; 37]. Similarly, vaccination with Hsp60 protected laboratory animals from the pulmonary infection caused by _P. brasiliensis_, suggesting this protein may also be found on the cell surface of this fungal species [66]. The _S. schenckii_ Hsp60 showed 99%, 96%, 96%, 96%, 95%, 91%, and 90% similarity to _S. brasiliensis_, _S. globosa_, _Ophisotoma piccae_, _Grosmannia clavigera_, _Sporothrix insectorum_, _H. capsulatus_, and _P. brasiliensis_ Hsp60, respectively. Moreover, it was predicted to contain 38 and 5 putative sites of O-linked and _N_-linked glycosylation, respectively, and no signal peptide was predicted. It was interesting to observe that six out of the 26 proteins identified in the PRM have an unknown obvious function, and we decided to work with the protein encoded by SPSK_00848, following the rationale that our results indicated a slightly higher sequence coverage percentage with this protein than that with the protein encoded by SPSK_05930. Additionally, when compared with the protein encoded by SPSK_04236, the protein encoded by SPSK_00848 (named here as Petidorhammomannan-associated protein 1, Pap1) contains 26 and five putative \(O\)-linked and \(N\)-linked glycosylation sites, values higher than those predicted for the SPSK_04236 product (22 and two putative \(O\)-linked and \(N\)-linked glycosylation sites, respectively). _S. schenckii_ Pap1 showed 79% similarity to _S. brasiliensis_ Pap1, but no obvious putative ortholog could be identified in _S.