Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

Kosalková Katarina, García-Estrada Carlos, Barreiro Carlos, Flórez, Jami, Paniagua, Martín

Description

The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

Sujets

• Chymosin
• Mold
• Vesicle

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 33
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Kosalková et al . Microbial Cell Factories 2012, 11 :5 http://www.microbialcellfactories.com/content/11/1/5

R E S E A R C H Open Access Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori . Proteomics studies reveal changes in the secretory pathway Katarina Kosalková 1 , Carlos García-Estrada 1 , Carlos Barreiro 1 , Martha G Flórez 2 , Mohammad S Jami 2 , Miguel A Paniagua 1 and Juan F Martín 1,2*

Abstract Background: The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results: Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important ‘ moonlighting ’ protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions: In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins. Keywords: secretory pathways, chymosin, filamentous fungi, casein phosphopeptides, vesicles, extracellular proteins

Background GRAS status in the food industry of several filamentous Filamentous fungi are very attractive host organisms for fungi such as Aspergillus niger, Aspergillus awamori, the production of heterologous proteins, since they have Aspergillus oryzae, Penicillium roqueforti among others several advantages for protein expression compared to [1,2], iii) rapid growth compared to other eukaryotic bacterial hosts. These advantages include i) the ability to cells, iv) the secretion of correctly folded functional pro- produce large amounts of extracellular proteins, ii) the teins and v) post-translational modifications, such as gly- cosylation. However, the levels of secreted heterologous artin ileon.es 1 * Fr C o or m re I s N p B o I n O d T e E n C, c I e: ns jf t . it m utod @ e u B n iotecnologíadeLeón,Avda.Realn°.1,Parque p n r e o c t k e s i i n n s t a h r e e s o e f c t r e e n to li ry mi p t a e t d h b w y ay p [ o 3 o , r 4 l ] y .I u n nd m e a r n st y o c o a d se b s, o l t i tl m e - - C F i u e ll n l t i í s fi t co of d a e ut L h e o ó r n i , n 2 fo 4 r 0 m 06 ati L o e n ó i n s , a S v p a a i i la n bleattheendofthearticl iting steps in the heterologous protein secretion occur e © 2012 Kosalková et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.