key: cord-0902503-89io82s4 authors: Xiao, Jing; Wang, Jinqiu; Cheng, Lei; Gao, Sihai; Li, Shugang; Qiu, Ning; Li, Hanmei; Peng, Lianxin; Geng, Fang title: A puzzle piece of protein N-glycosylation in chicken egg: N-glycoproteome of chicken egg vitelline membrane date: 2020-08-26 journal: Int J Biol Macromol DOI: 10.1016/j.ijbiomac.2020.08.193 sha: 9c7c5c3c85bc79d8d2d0eb72dac2fbbc340172a8 doc_id: 902503 cord_uid: 89io82s4 The chicken egg vitelline membrane (CEVM) is an important structure for the transmembrane transport of egg yolk components, protection of the blastodisc, and separation of egg white and egg yolk. In this study, the N-glycoproteome of the CEVM was mapped and analyzed in depth. Total protein of the CEVM was digested, and the glycopeptides were enriched by a hydrophilic interaction liquid chromatography microcolumn and identified by nano liquid chromatography/tandem mass spectrometry. A total of 435 N-glycosylation sites on 208 N-glycoproteins were identified in CEVM. Gene Ontology enrichment analysis showed that CEVM N-glycoproteins are mainly involved in the regulation of proteinases/inhibitors and transmembrane transport of lipids. Mucin-5B is the primary N-glycoprotein in the CEVM. Comparison of the main N-glycoproteins between the CEVM and other egg parts revealed the tissue specificity of N-glycosylation of egg proteins. The results provide insights into protein N-glycosylation in the chicken egg, CEVM functions and underlying mechanisms. transport of egg yolk material. The CEVM is the most important and most complex part of the chicken egg despite being thin (approximately 10 μm) and accounting for only for a small part (2%~3%) of the total weight of the egg. The in-depth exploration of the composition of the CEVM will help reveal its functions and the underlying mechanisms. Similar to egg white, the CEVM has a water content of 88%, and protein accounts for 87% of its dry matter. However, the protein profile of the CEVM is very different from that of egg white. In a previous CEVM proteome analysis, a total of 137 proteins were identified, and the relative abundance of CEVM proteins was estimated. The high-abundance CEVM proteins included some egg white proteins, such as ovalbumin, lysozyme, ovotransferrin, ovalbumin-related protein Y, and ovomucin, and the main proteins in egg yolk, such as apolipoprotein D and vitellogenin-2. Among the low-abundance CEVM proteins, some eggshell matrix-specific proteins were found, such as ovocleidin-17, ovocleidin-116, and ovocalyxin-36. Vitelline outer-membrane proteins and zona pellucida-related proteins were also major components of the CEVM [1] . These results reveal the diversity of protein sources in CEVM. However, our understanding of CEVM proteins remains incomplete. For example, the posttranslational modification of CEVM proteins has not been studied. a proteinase/inhibitor regulation system and complement/immune systems in chicken egg yolk [3] . Subsequent studies of duck and quail egg N-glycoproteomes revealed differences in N-glycosylation between species and provided insight into evolution and environmental adaptability [4] [5] [6] . Furthermore, a quantitative glycoproteome analysis of unhatched fertilized eggs and hatched eggs over a 12-day period indicated that N-glycoprotein variations affected the utilization of egg proteins by the chicken embryo during incubation [7] . These studies provided important structural information about egg N-glycoproteins and enhanced our understanding of the mechanisms of innate defense and embryo protection, egg protein functions, evolution, embryo development, and changes in egg characteristics during storage and processing. The study of the CEVM N-glycoproteome is expected to clarify the mechanisms underlying CEVM functions. Therefore, the present study aimed to characterize and analyze the CEVM N-glycoproteome. The total proteins of the CEVM were extracted and digested, and the glycopeptides were enriched by hydrophilic interaction liquid chromatography and subsequently deglycosylated by using PNGase F in H 2 18 O. The -18 OH-labeled deglycopeptides were then identified by nano liquid chromatography/tandem mass spectrometry. In addition, bioinformatics analysis was employed to elucidate the functions of CEVM N-glycoproteins and the associated biological processes. Ecological Food Co., Ltd. (Mianyang, Sichuan) within 24 h of laying and used in this study [8] [9] [10] . The eggshells were broken, and the egg yolks were rolled on filter paper to remove the egg white [3, 9] . A pipet tip was used to penetrate the CEVM; after the yolk flowed out, the CEVM was washed using PBS buffer (10 mmol/L, pH 7.2, containing 0.14 mol/L NaCl). The CEVMs were rinsed in PBS buffer and magnetically stirred (30 r/min) 5 times for 30 min each time. Twelve CEVMs were mixed as one biological replicate, and a total of 3 biological replicates were produced. The collected CEVM samples were frozen and stored in liquid nitrogen until further analysis. Total protein was extracted according to previous methods [3, 11] . Briefly, the frozen CEVM samples (one biological replicate consisting of 12 CEVMs) were ground with liquid nitrogen into powder. Then, the powder was transferred into a 5-mL centrifuge tube, and lysis buffer (8 mol/L urea, 1% protease inhibitor cocktail) was added at a mass ratio of 1:4 (sample:buffer proteins in the species database were set as the background. GO terms with a corrected p <0.05 were considered significant. The CEVM proteins were extracted and digested, and the CEVM glycopeptides Table S1 ). The mass error of all peptide ions was less than 5 ppm ( Mucin-5B has also been identified in N-glycoproteomic analyses of egg white (with 15 N-glycosites) and chalaza (with 34 N-glycosites) [2] . However, mucin-5B is absent from the N-glycoproteomes of egg yolk and eggshell matrix, indicating that the distribution of mucin-5B is tissue specific [3, 10] . In the CEVM, mucin-5B was identified with 28 N-glycosites and a total "MS/MS count" of 564. Interestingly, the N-glycosite locations of mucin-5B from different egg parts are quite different (Fig. 3 ). There may be several reasons for this difference. 1) The analysis method differed among studies. 2) The relative content of mucin-5B differs among egg parts, and a higher relative content would generally be beneficial for improving the depth of in an N-glycoproteomic analysis of chicken egg white [2] . In the present study, in addition to these 4 sites previously identified, 3 new N-glycosites (N 387 , N 959 , and N 1020 ) of ovomucin were found in the CEVM. The highly glycosylated ovomucin in egg white is considered the main reason for the high viscosity of egg whites, and ovomucin undergoes disaggregation during storage, accompanied by the thinning of egg white [17] . Furthermore, it was previously found that the abundances of mucin-5B and mucin-6 in thick egg white were 18.9% and 400.0% higher, respectively, than those in thin egg white (p < 0.01). The higher content of mucin-5B/mucin-6 in thick egg white could provide more skeleton structure during the formation of heat-induced gel and might regulate the microstructure of the gel, resulting in a "softer and tougher" gel texture [18] . These findings suggest that the abundance of ovomucin is related to the viscosity and texture properties of egg white. Therefore, it could be inferred that the high abundances of mucin-5B and mucin-6 in the CEVM might enhance CEVM mechanical strength by increasing its viscosity and toughness. Similar to the thinning of egg white, the decrease in mechanical strength of CEVM during storage might also related to the disaggregation of ovomucin. This is a important speculation that needs to be verified by future study. Ovomucin (mucin-5B and mucin-6) has been identified as absent from egg yolk in all previous proteome studies [3, 5, 19, 20] , suggesting that ovomucin is Eggshell membrane has been developed as wound healing materials [22, 23] . Compared with eggshell membrane, CEVM has potential advantages due to the adhesion and antiviral properties brought by high abundance of ovomucin. In addition, ovomucin could provide an important innate immune barrier against various toxins and pathogens, and the sialic acid at the end of the glycans is essential in the recognition of avian influenza viruses [24] [25] [26] . Therefore, the high abundance of ovomucin in the CEVM provides a strong natural defense, protecting the chicken embryo (or preserving the egg yolk). Moreover, the diversity and heterogeneity of N-glycosylation sites of ovomucin may be related to the efficient recognition of influenza virus by this protein. Alpha-2-macroglobulin-like protein 1 (A2ML1) was identified in the CEVM Typical members of this family are glycoproteins composed of four identical subunits with high molecular weight. The primary function of alpha-2-macroglobulins is the inhibition of various proteases by encapsulating them in their cage-like molecules [27] . Alpha-2-macroglobulins are involved in innate immunity [28] , tumor development [29] , cell migration [30] , and other processes. These observations suggest that the high abundance of A2ML1 in the CEVM might have important functions such as inhibiting proteases secreted by microorganisms to resist invasion and maintaining the balance of proteases on the CEVM to maintain the integrity of the membrane structure. Apolipoprotein B is the precursor protein of egg yolk low-density lipoprotein, and it was previously identified as the predominant glycoprotein in egg yolk, with a total of 35 N-glycosites [3] . In the present study, apolipoprotein B was similarly the Previous investigations have shown that the HEP21 gene is expressed primarily in hen oviduct, especially the magnum, where egg white proteins are secreted [33] . Egg proteomic studies have shown that at the protein expression level, HEP21 is present in all parts of the egg, including the egg white, egg yolk, eggshell matrix, chalaza, and CEVM. As for the N-glycosylation level, N-glycosylated HEP21 has been found in all egg parts except egg white. In the present study, 3 N-glycosites of The function of HEP21 remains unclear, but research has shown that the expression of HEP21 mRNA is responsive to estrogen. Therefore, HEP21 is thought to play a regulatory role in the production and formation of the egg [34] . Hormone signaling and target-tissue responses to hormones need to be precise. Therefore, as a protein involved in estrogen responses, the tissue specificity of HEP21 at different levels (gene expression, protein expression and N-glycosylation) can be expected to greatly enhance the diversity of specific responses, thereby facilitating accurate response to estrogen. Clusterin was identified in egg white and eggshell in previous N-glycoproteome analyses and in CEVM in the present study; however, it has not been identified in the N-glycoproteome of egg yolk. A total of 4 N-glycosites (N 99 , N 141 , N 352 , and N 372 ) of CEVM clusterin were identified, all of which have been found to be N-glycosylated in egg white and eggshell clusterin. In the previous N-glycoproteomic analysis of egg white, heterogeneity of clusterin glycosylation was revealed by two-dimensional electrophoresis, and glycosylation was found to strongly influence the charge characteristics (isoelectric point) of clusterin [2] . 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