1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 ABSTRACT: Chitotriosidase (HCHT) is one of two family 18 chitinases produced by 45 humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be 46 part of the human defense mechanism against fungal parasites, but its precise role and the 47 details of its enzymatic properties have not yet been fully unraveled. We have studied 48properties of HCHT by analyzing how the enzyme acts on high molecular-weight chitosans, 49 soluble co-polymers of β-1,4-linked N-acetylglucosamine (GlcNAc, A) and glucosamine 50 (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial 51 family 18 enzymes, we show that HCHT degrades chitosan primarily via an endo-processive 52 mechanism, as would be expected on the structural features of its substrate-binding cleft. The 53 preferences of HCHT subsites for acetylated versus non-acetylated sugars were assessed by 54 sequence analysis of obtained oligomeric products showing a very strong, absolute, and a 55 relative weak preference for an acetylated unit in the -2, -1, +1 subsite, respectively. The 56 latter information is important for the design of inhibitors that are specific for the human 57 chitinases and also provide insight into what kind of products may be formed in vivo upon 58 administration of chitosan-containing medicines or food products. 59 KEYWORDS: Human chitinase; chitosan; chitin; processivity; chitotriosidase. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 Chitin, an insoluble linear polysaccharide consisting of repeated units of β-1,4-N-66 linked acetylglucosamine [(GlcNAc) n ], is common as a structural polymer in crustaceans, 67 arthropods, fungi, and parasitic nematodes. The metabolism of chitin in nature is controlled 68 by enzymatic systems that produce and break down chitin, primarily chitin synthases and 69 chitinases, respectively. Chitinases are thought to play important roles in anti-parasite 70 responses in several life forms, including humans (1-4). Even though chitin and chitin 71 synthases have not been found in humans, we produce two active chitinases that are 72 categorized as family 18 chitinases based on sequence-based classification of glycoside 73 hydrolases (5). These two enzymes are called acidic mammalian chitinase (AMCase) (6) and 74 human chitotriosidase (HCHT) (7) and both are believed to play roles in anti-parasite 75 responses (8, 9). While AMCase is found in the stomach (6), in tears (10), sinus mucosa (11), 76 and lungs (12), HCHT is primarily expressed in activated human macrophages (13). 77HCHT is up-regulated in a series of diseases and medical conditions such as 78Ga...

a b s t r a c tHuman chitotriosidase (HCHT) is a family 18 chitinase that is an innate part of the immune system. We have mapped preferred productive binding modes of chito-oligosaccharide substrates to HCHT and the data show that HCHT has strong binding affinity in the +3 subsite. Moreover, HCHT shows anomer-specific binding affinities in subsites +2 and +3. These features could endorse HCHT with higher endo-activity and a higher transglycosylation potential.