A new study by researchers from the University of Piemonte Orientale – Italy and Sant’Andrea Hospital – Italy has found that SARS-CoV-2 infections leads to alterations of the circulating peptidome.
The study findings were published in the peer reviewed International Journal of Environmental Research and Public Health.
The comparison between the peptidome profile of SARS-CoV-2 positive and negative patients reported the quantitative modulation of 73 peptides from 31 different proteins, the majority of which were downregulated in COVID-19 infected patients (fold change < 0.5). Of those peptides, 14 were from albumin and their decrease is in line with the well-documented decrease in albumin levels in COVID-19 patients .
In addition, the decrease of apolipoprotein A-1 derived peptides (10 peptides) is in accordance with the reported decrease of circulating HDL . Furthermore, for apolipoprotein C-III the total levels were reported to be dysregulated in relation to COVID-19, confirming the observed changes in the degradation pattern with an increase of cutting efficiency at position 28 that justifies the increase of S21EAEDASL28 peptide, and the concomitant decrease of 21–84 and 21–34 fragments upon infection [41,42].
Despite the well-known correlation between apolipoprotein levels and metabolic disorders, the design of the present study did not allow it. In fact, among patients involved in the research, just a few reported metabolic disorders, thus limiting the possibility of identifying potential correlations between apolipoproteins and metabolic diseases. A further study designed properly could contribute to the understanding of this potential relationship.
Other previous studies performed on healthy subjects identified a larger number of peptides, but these were mainly limited to collagen type I alpha 1 chain, fibrinogen alpha chain, alpha-1 antitrypsin, and thymosin β-4 proteins. These differences are mainly due to the different isolation and analytical methods that were employed by the authors [43,44].
Indeed, we took advantage of a micro liquid chromatography system for our analysis, certainly less sensitive compared to other analytical techniques (nanoLC system, CE-MS), but more reproducible and able to guarantee a high throughput approach. Moreover, our analysis partially overlapped the results obtained in previous works; in addition to peptides deriving from proteins commonly found in plasma samples (transport proteins, enzyme inhibitors, complement factors, etc.), we were able to identify some unique and less abundant peptides (Retinol-binding protein, Plasminogen, Immunoglobulin gamma-1 heavy chain).
Moreover, in accordance with our analysis, a number of peptides deriving from proteins associated with the inflammatory process are decreased in SARS-CoV-2 infected patients: for example, human alpha-1-antitrypsin (A1AT_HUMAN), human inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2_HUMAN), human complement C3 (CO3_HUMAN), and human complement C1q subcomponent subunit B (C1QB_HUMAN) peptides.
Surprisingly, although alpha-1-antitrypsin and inter-alpha-trypsin inhibitors are reported to increase during COVID-19 (20), the derived peptides are decreasing in our analysis (5 concordant peptides for both). This may indicate enhanced stability or escape from degradation may contribute to the increase of circulating levels.
Human alpha-1-antitrypsin (A1AT) is a circulating protease inhibitor mainly synthesized by hepatocytes and targeting neutrophil elastase, whose activity is critical for the prevention of proteolytic tissue (lung and liver) damage . It has been enlightened the dual role of A1AT as an antiviral and anti-inflammation protein.
Indeed, alpha-1-antitrypsin not only can protect the lung from damages such as emphysema by inhibiting neutrophilic elastase (anti-inflammation role) but can also block SARS-CoV-2 infection through the inhibition of a protease involved in the entry of SARS-CoV-2 into the host cells (transmembrane protease serine-type 2-TMPRSS2), thus exerting an antiviral action [46,47].
Immune dysregulation and the more severe symptoms shown by COVID-19 patients may be so associated with alterations in A1AT levels.
In the same way, inter-alpha-trypsin inhibitors are an acute phase protein family with matrix protective activity through protease inhibitory action. Since previous studies pointed out that low levels of inter-alpha-trypsin inhibitor proteins correlated with severe sepsis and influenced patient survival [48,49], these observations may help explain the downregulation of human inter-alpha-trypsin inhibitor heavy chain H2 peptide in SARS-CoV-2 infected subjects, when compared to negative controls.
Interestingly, the complement system fragments were under-expressed in positive subjects. As diffuse complement activation is one of the key features of COVID-19, a variation of complement factors derived peptides is not surprising. We observed complement C1q and C3 fragments (respectively, 1 and 6 peptides) decreasing during infection, and C4 changing digestion pattern with 1 peptide downregulated and 3 upregulated.
Even if the involvement of the humoral immune response in COVID-19 development has been largely demonstrated, the role of various complement system components in the prognosis of SARS-CoV-2 infection remains nevertheless unclear . According to Wu et al. , who investigated the clinical and immunological characteristics of COVID-19 patients stratified on the basis of disease severity, complement C1q subcomponent subunit B levels were, for instance, significantly reduced in severe cases.
Conversely, complement C3 levels have been either associated with poor prognosis in severe patients [52,53] or addressed as not predictive of disease progression . It can be assumed that the alterations in the degradation pattern of the considered proteins correlate with the hyperactivation of the complement pathway already demonstrated in SARS-CoV-2 infection.
In contrast, the comparison of COVID-19 positive subjects vs. negative controls also established the upregulation of only a few peptides: more specifically, transthyretin (TTHY_HUMAN), isoform 2 of Haptoglobin (HPT_HUMAN), Ceruloplasmin (CERU_HUMAN), isoform 2 of Fibrinogen alpha chain (FIBA_HUMAN), and isoform 2 of Complement C4-A (CO4A_HUMAN) peptides.
Transthyretin is an acute phase-reactant acting as a hormone transporter whose levels negatively correlate with inflammation, so much that low concentrations of TTHY are indicative of a systemic inflammatory state .
Our analysis, in keeping with what we have just reported, highlights a decrease in transthyretin protein levels; our data suggest a parallel change in degradation with an increase cutting at position 137 indicated by increased S137TTAVVTNPKE147 and the concomitant decrease of 129–147, 130–147, and 116–147 fragments.
Although transthyretin protein has been proposed as a marker of nutritional status, the potential contribution of the feeding status in the modulation of identified peptides was excluded . In fact, TTHY is a marker of malnutrition and of prognosis associated with malnutrition, and, although dietary questionnaires were not available, no malnourished patients were reported in the clinical data.
In addition, all the samples were obtained at the diagnosis of the infection, as soon as the patients were recovered at the hospital, thus excluding any impact of the therapy and the disease on nutrition. However, further studies are needed to definitely exclude the contribution of feeding status.
Severe COVID-19 patients often exhibit signs of hemolysis and consequently elevated levels of free heme, whose excess promotes oxidative and inflammatory stress. The acute phase protein haptoglobin (Hpt) can scavenge free heme, preventing heme-induced inflammation, but in the case of severe hemolysis, this protein may be overwhelmed, hence not completely neutralizing heme’s dangerous effects .
The observed upregulation of HPT_HUMAN peptide in mild to severe SARS-CoV-2 patients, therefore, led us to postulate that infected subjects show high levels of the related protein, which is unable to efficiently neutralize free heme. Moreover, the presence of an upregulated peptide together with two downregulated ones strongly suggests a change in the degradation pattern.
Similarly, ceruloplasmin (Cp), positive acute phase reactant, and the coagulation factor fibrinogen are known key molecular players in inflammation [58,59]. For this reason, it is not surprising to notice an increase in the corresponding peptide levels and of the whole proteins as well in COVID-19 positive patients.
Lastly, the modulation of complement C4-A peptide in SARS-CoV-2 infected subjects is consistent with the changes in complement pathway activation stated before.
The lists of severe to negative and mild to negative peptides significantly overlap with the previous data, with the interesting exception of a peptide derived from the cytoskeletal protein talin. This peptide is specific for severe patients and one of the features discriminating mild to severe ones. Other differences are instead quantitative, with a stronger upregulation of transthyretin, modulation of haptoglobin peptides, and downregulation of fibrinogen, complement, and alpha-1-antitrypsin in severe versus mild cases.
The broad spectrum of the COVID-19 symptoms seems to be attributable to a variety of pathophysiological mechanisms. Among them, matrix metalloproteinases (MMPs), a family of zinc-dependent and Ca2+-containing endoproteinases deriving from inflammatory and parenchymal cells in the lung, have been already reported as involved in pulmonary pathologies characterized by tissue remodeling and acute lung injury (e.g., asthma, COPD) .
Despite the involvement of protease activity in COVID-19, it is not easy to link peptide abundance to changes in the activity of specific proteases. A search in the MEROPS protease database  indicates that the proteases responsible for the generation of the above-mentioned peptides are largely unknown.
A prediction based on the cleavage sites indicates the metalloproteinase family, catepsin family, meprin A, pepsin A-3, and calpain1/2 as proteases generating the regulated peptides. Changes in protease activity are indeed expected in an inflammatory disease with strong neutrophil and complement activation . In particular, a strong increase in matrix-metalloproteinase 9 (MMP-9), whose correlation with acute lung injury and chronic respiratory diseases is a fact, has been reported in COVID-19 patients and associated with respiratory failure .
In the same way, a correlation between MMP-3 serum levels and the severity of COVID-19 pulmonary symptoms has been also assessed . Thus, given the observed alterations in the degradation pattern of the cited proteins and the reported involvement of metallopeptidases in SARS-CoV-2 infection, there may be a link between these two phenomena impacting COVID-19 severity.
However, it must be noted that the changes in the circulating peptidome observed are the result of a complex balance between target abundance, protease activity, and clearance rate, all of which could be modulated by COVID-19.