Diversity of human salivary heparan sulfate

Abstract

The human oral cavity and upper airway serves as an early barrier and reservoir in the transmission of SARS-CoV-2. Saliva in this microenvironment may serve as a key host factor that can modulate susceptibility to infection and eventual infection of the lower respiratory tract. We sought to analyze the content and composition of heparan sulfate, a glycosaminoglycan identified as an important co-receptor for viral entry, and whether there is any correlation with SARS-CoV-2 infection. We enlisted 98 participants stratified by age, gender, race, and COVID-19 history. Notably, the concentration of heparan sulfate in saliva increased with age, and its composition showed a wide range of variability within each age group independently of age. Heparan sulfate concentration and composition did not differ significantly with gender, ethnicity or race. Compared to patients with no COVID-19 history, patients with previous infection had a similar salivary heparan sulfate concentration, but significant increases in overall sulfation were noted. Moreover, in a subset of participants, for which data was available pre- and post- infection, significant elevation in N-sulfoglucosamine in heparan sulfate was observed post- COVID-19. Examination of salivary bacterial 16S rRNA, showed a significant reduction in species predicted to possess heparan sulfate-modifying capacity among participants >60 years old, which correlates with the increase in heparan sulfate content in older individuals. These findings demonstrate a surprisingly wide variation in heparan sulfate content and composition in saliva across the sampled population and confirm other findings showing variation in content and composition of glycosaminoglycans in blood and urine.

Keywords: Covid-19; age; heparan sulfate; microbiome; saliva.

Fig. 1

Variation in the concentration of salivary heparan sulfate across different age groups. Heparan sulfate was purified from 3 mL of saliva from each donor, digested with heparin lyases, and the resulting disaccharides were tagged with aniline. The heparan sulfate disaccharides were quantified by liquid chromatography-mass spectrometry and normalized to the volume of sample. The donors were divided into 4 groups based on age to achieve approximate equal distribution; (A) group 1: 7–17 years old, (B) group 2: 18–40 years old, (C) group 3: 41–60 years old, and (D) group 4: 61–91 years old. Each dataset is sorted by donor age in ascending order.

Fig. 2

Distribution of each outcome by age. The distribution of the salivary heparan sulfate concentration data presented in Fig. 1 and Table 1 was determined for each age group and the total sample population. The boxplot demonstrates the trend towards increasing concentration of salivary heparan sulfate with age.

Fig. 3

Variation in non-sulfated and monosulfated disaccharides across different age groups. The relative content of non-sulfated (D0S0) and monosulfated disaccharides (D0S0 and D0A6) was determined for each age group. The data is sorted by donor age in ascending order. The disaccharide structure code is used: D0A0, ΔUA-GlcNAc; D0S0, ΔUA-GlcNS; D0A6, ΔUA-GlcNAc6S; where ΔUA is 4,5-unsaturated uronic acid (7).

Fig. 4

Variation in disulfated and trisulfated disaccharides in different age groups. The relative content of disulfated (D0S6, D2S0) and trisulfated disaccharides (D2S6) was determined for each age group. Each dataset is sorted by donor age in ascending order. The disaccharide structure code is used: D0S6, ΔUA-GlcNS6S; D2S0, ΔUA2S-GlcNS; D2S6, ΔUA2S-GlcNS6S; where ΔUA is 4,5-unsaturated uronic acid (7).

Fig. 5

The log-ratio of predicted heparan sulfate-modifying microbial species to other microbial species. DNA was extracted from saliva samples and analyzed by 16S sequencing. The number of microbial species with the capacity to degrade or modify heparan sulfate (heparan sulfate spp.) relative to those with no predicted capacity (other spp.) was analyzed across the individuals in the different age groups.