Neil N. Trivedi

Mast Cell Peptidases: Chameleons of Innate Immunity and Host Defense

Mast cells make and secrete an abundance of peptidases, which are stored in such large amounts in granules that they comprise a high fraction of all cellular protein. Perhaps no other immune cell is so generously endowed with peptidases. For many years after the main peptidases were first described, they were best known as markers of degranulation, for they are released locally in response to mast cell stimulation and can be distributed systemically and detected in blood. The principal peptidases are tryptases, chymases, carboxypeptidase A3, and dipeptidylpeptidase I (cathepsin C). Numerous studies suggest that these enzymes are important and even critical for host defense and homeostasis. Endogenous and allergen or pathogen-associated targets have been identified. Belying the narrow notion of peptidases as proinflammatory, several of the peptidases limit inflammation and toxicity of endogenous peptides and venoms. The peptidases are interdependent, so that absence or inactivity of one enzyme can alter levels and activity of others. Mammalian mast cell peptidases--chymases and tryptases especially--vary remarkably in number, expression, biophysical properties, and specificity, perhaps because they hyper-evolved under pressure from the very pathogens they help to repel. Tryptase and chymase involvement in some pathologies stimulated development of therapeutic inhibitors for use in asthma, lung fibrosis, pulmonary hypertension, ulcerative colitis, and cardiovascular diseases. While animal studies support the potential for mast cell peptidase inhibitors to mitigate certain diseases, other studies, as in mice lacking selected peptidases, predict roles in defense against bacteria and parasites and that systemic inactivation may impair host defense.

Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number

Elevated basal serum tryptase levels are present in 4–6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase–encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.

How Immune Peptidases Change Specificity: Cathepsin G Gained Tryptic Function but Lost Efficiency during Primate Evolution

Cathepsin G is a major secreted serine peptidase of neutrophils and mast cells. Studies in Ctsg-null mice suggest that cathepsin G supports antimicrobial defenses but can injure host tissues. The human enzyme has an unusual “Janus-faced” ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu226 in the primary specificity pocket and underlies proposed important functions, such as activation of prourokinase. However, most mammals, including mice, substitute Ala226 for Glu226, suggesting that human tryptic activity may be anomalous. To test this hypothesis, human cathepsin G was compared with mouse wild-type and humanized active site mutants, revealing that mouse primary specificity is markedly narrower than that of human cathepsin G, with much greater Tyr activity and selectivity and near absence of tryptic activity. It also differs from human in resisting tryptic peptidase inhibitors (e.g., aprotinin), while favoring angiotensin destruction at Tyr4 over activation at Phe8. Ala226Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate prourokinase. Phylogenetic analysis reveals that the Ala226Glu missense mutation appearing in primates 31–43 million years ago represented an apparently unprecedented way to create tryptic activity in a serine peptidase. We propose that tryptic activity is not an attribute of ancestral mammalian cathepsin G, which was primarily chymotryptic, and that primate-selective broadening of specificity opposed the general trend of increased specialization by immune peptidases and allowed acquisition of new functions.

Mast Cell α and β Tryptases Changed Rapidly during Primate Speciation and Evolved from γ-Like Transmembrane Peptidases in Ancestral Vertebrates

Human mast cell tryptases vary strikingly in secretion, catalytic competence, and inheritance. To explore the basis of variation, we compared genes from a range of primates, including humans, great apes (chimpanzee, gorilla, orangutan), Old- and New-World monkeys (macaque and marmoset), and a prosimian (galago), tracking key changes. Our analysis reveals that extant soluble tryptase-like proteins, including α- and β-like tryptases, mastins, and implantation serine proteases, likely evolved from membrane-anchored ancestors because their more deeply rooted relatives (γ tryptases, pancreasins, prostasins) are type I transmembrane peptidases. Function-altering mutations appeared at widely separated times during primate speciation, with tryptases evolving by duplication, gene conversion, and point mutation. The α-tryptase Gly216Asp catalytic domain mutation, which diminishes activity, is present in macaque tryptases, and thus arose before great apes and Old World monkeys shared an ancestor, and before the αβ split. However, the Arg−3Gln processing mutation appeared recently, affecting only human α. By comparison, the transmembrane γ-tryptase gene, which anchors the telomeric end of the multigene tryptase locus, changed little during primate evolution. Related transmembrane peptidase genes were found in reptiles, amphibians, and fish. We identified soluble tryptase-like genes in the full spectrum of mammals, including marsupial (opossum) and monotreme (platypus), but not in nonmammalian vertebrates. Overall, our analysis suggests that soluble tryptases evolved rapidly from membrane-anchored, two-chain peptidases in ancestral vertebrates into soluble, single-chain, self-compartmentalizing, inhibitor-resistant oligomers expressed primarily by mast cells, and that much of present numerical, behavioral, and genetic diversity of α- and β-like tryptases was acquired during primate evolution.

Chimerism, point mutation, and truncation dramatically transformed mast cell δ-tryptases during primate evolution

BACKGROUND Tryptases are serine peptidases stored in mast cell granules. Rodents express 2 soluble tryptases, mast cell proteases (MCPs) 6 and 7. Human alpha- and beta-tryptases are orthologs of MCP-6. However, much of the ancestral MCP-7 ortholog was replaced by parts of other tryptases, creating chimeric delta-tryptase. Human delta-tryptases limited activity is hypothesized to be due to truncation and processing mutations. OBJECTIVE We sought to probe the origins and consequences of mutations in primate delta-tryptases. METHODS Prosimian (lemur), monkey (macaque), great ape (orangutan, gorilla, and chimpanzee), and human delta-tryptase genes were identified by means of data mining and genomic sequencing. Resulting genes were analyzed phylogenetically and structurally. RESULTS The seminal conversion event generating the delta-tryptase chimera occurred early because all primates studied contain delta-tryptase genes. Truncation, resulting from a nonsense mutation of Trp206, occurred much later, after orangutans and other great apes last shared an ancestor. The Arg-3Gln propeptide mutation occurred most recently, being present in humans and chimpanzees but not in other primates. Surprisingly, the major active tryptase in monkeys is full-length delta-tryptase, not beta-tryptase, which is the main active tryptase in human subjects. Models of macaque delta-tryptase reveal that the segment truncated in human subjects contains antiparallel beta-strands coursing through the substrate-binding cleft, accounting for truncations drastic effect on activity. CONCLUSIONS Transformations in the ancestral MCP-7-like gene during primate evolution caused dramatic variations in function. Although delta-tryptases are nearly inactive in humans, they are active and dominant in monkeys.

Guinea pig chymase is leucine-specific: a novel example of functional plasticity in the chymase/granzyme family of serine peptidases.

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the α clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig α-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by α1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster α-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.

Phenotypic variability in human skin mast cells

Mast cells (MCs) are unique constituents of the human body. While inter‐individual differences may influence the ways by which MCs operate in their skin habitat, they have not been surveyed in a comprehensive manner so far. We therefore set out to quantify skin MC variability in a large cohort of subjects. Pathophysiologically relevant key features were quantified and correlated: transcripts of c‐kit, FcεRIα, FcεRIβ, FcεRIγ, histidine decarboxylase, tryptase, and chymase; surface expression of c‐Kit, FcεRIα; activity of tryptase, and chymase; histamine content and release triggered by FcεRI and Ca2+ ionophore. While there was substantial variability among subjects, it strongly depended on the feature under study (coefficient of variation 33‐386%). Surface expression of FcεRI was positively associated with FcεRIα mRNA content, histamine content with HDC mRNA, and chymase activity with chymase mRNA. Also, MC signature genes were co‐regulated in distinct patterns. Intriguingly, histamine levels were positively linked to tryptase and chymase activity, whereas tryptase and chymase activity appeared to be uncorrelated. FcεRI triggered histamine release was highly variable and was unrelated to FcεRI expression but unexpectedly tightly correlated with histamine release elicited by Ca2+ ionophore. This most comprehensive and systematic work of its kind provides not only detailed insights into inter‐individual variability in MCs, but also uncovers unexpected patterns of co‐regulation among signature attributes of the lineage. Differences in MCs among humans may well underlie clinical responses in settings of allergic reactions and complex skin disorders alike.

Bronchoalveolar Lavage Cell Immunophenotyping Facilitates Diagnosis of Lung Allograft Rejection

Supplementary methods to identify acute rejection and to distinguish rejection from infection may improve clinical outcomes for lung allograft recipients. We hypothesized that distinct bronchoalveolar lavage (BAL) cell profiles are associated with rejection and infection. We retrospectively compared 2939 BAL cell counts and immunophenotypes against concomitantly obtained transbronchial biopsies and microbiologic studies. We randomly assigned 317 subjects to a derivation or validation cohort. BAL samples were classified into four groups: infection, rejection grade ≥A1, both or neither. We employed generalized estimating equation and survival modeling to identify clinical predictors of rejection and infection. We found that CD25+ and natural killer cell percentages identified a twofold increased odds of rejection compared to either the infection or the neither infection nor rejection groups. Also, monocytes, lymphocytes and eosinophil percentages were independently associated with rejection. A four‐predictor scoring system had high negative predictive value (96–98%) for grade ≥A2 rejection, predicted future rejection in the validation cohort and predicted increased risk of bronchiolitis obliterans syndrome in otherwise benign samples. In conclusion, BAL cell immunophenotyping discriminates between infection and acute rejection and predicts future outcomes in lung transplant recipients. Although it cannot replace histopathology, immunophenotyping may be a clinically useful adjunct.

Mutational Tail Loss Is an Evolutionary Mechanism for Liberating Marapsins and Other Type I Serine Proteases from Transmembrane Anchors

Background: Vertebrate marapsins can be either type I transmembrane proteases or unanchored. Results: Point mutations liberated marapsins from transmembrane peptides independently in human-related primates and other mammalian clades. Soluble marapsins are active and inhibitor-resistant. Conclusion: Mutational tail loss transformed transmembrane marapsins and related proteins into soluble proteases. Significance: These findings suggest a general evolutionary mechanism for evolving proteases with new properties and functions. Human and mouse marapsins (Prss27) are serine proteases preferentially expressed by stratified squamous epithelia. However, mouse marapsin contains a transmembrane anchor absent from the human enzyme. To gain insights into physical forms, activities, inhibition, and roles in epithelial differentiation, we traced tail loss in human marapsin to a nonsense mutation in an ancestral ape, compared substrate preferences of mouse and human marapsins with those of the epithelial peptidase prostasin, designed a selective substrate and inhibitor, and generated Prss27-null mice. Phylogenetic analysis predicts that most marapsins are transmembrane proteins. However, nonsense mutations caused membrane anchor loss in three clades: human/bonobo/chimpanzee, guinea pig/degu/tuco-tuco/mole rat, and cattle/yak. Most marapsin-related proteases, including prostasins, are type I transmembrane proteins, but the closest relatives (prosemins) are not. Soluble mouse and human marapsins are tryptic with subsite preferences distinct from those of prostasin, lack general proteinase activity, and unlike prostasins resist antiproteases, including leupeptin, aprotinin, serpins, and α2-macroglobulin, suggesting the presence of non-canonical active sites. Prss27-null mice develop normally in barrier conditions and are fertile without overt epithelial defects, indicating that marapsin does not play critical, non-redundant roles in development, reproduction, or epithelial differentiation. In conclusion, marapsins are conserved, inhibitor-resistant, tryptic peptidases. Although marapsins are type I transmembrane proteins in their typical form, they mutated independently into anchorless forms in several mammalian clades, including one involving humans. Similar pathways appear to have been traversed by prosemins and tryptases, suggesting that mutational tail loss is an important means of evolving new functions of tryptic serine proteases from transmembrane ancestors.